Compositions comprising fibrous polypeptides and polysaccharides

ABSTRACT

Isolated polypeptides are disclosed comprising an amino acid sequence encoding a monomer of a fibrous polypeptide attached to a heterologous polysaccharide binding domain. Composites comprising same, methods of generating same and uses thereof are all disclosed.

RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.13/870,032 filed on Apr. 25, 2013, which is a division of U.S. patentapplication Ser. No. 12/744,703 filed on May 26, 2010, now U.S. Pat. No.8,431,158, which is a National Phase of PCT Patent Application No.PCT/IL2008/001542 filed on Nov. 26, 2008, which claims the benefit ofpriority under 35 USC §119(e) of U.S. Provisional Patent ApplicationNos. 61/071,968 filed on May 28, 2008 and 60/996,581 filed on Nov. 26,2007. The contents of the above applications are incorporated byreference as if fully set forth herein in their entirety.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 61120SequenceListing.txt, created on Dec. 8,2014, comprising 127,318 bytes, submitted concurrently with the filingof this application is incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates tocompositions comprising fibrous polypeptides and polysaccharides anduses of same.

The most extensively investigated biological polymers for use inmaterial science are polysaccharides due to their abundance andextremely diverse mechanical properties.

The polysaccharide cellulose is the most common biopolymer on earth.Although it is mostly found in plant biomass, it is also produced byanimals, fungi and bacteria. Cellulose is a crystalline assembly ofcellobiose subunits which are made from glucose. Due to its crystallinestructure, cellulose has high tensile strength and elasticityapproaching that of synthetic carbon fibers, and it has a very favorablestrength/weight ratio compared to, for example, steel. In plant cellwalls, cellulose is found as a composite with other polysaccharides suchas hemicellulose, pectin, lignin, enzymes and structural proteins. Thesemolecules link the cellulose microfibrils improving the mechanism ofload transfer when the cell is subjected to mechanical stress whilstenhancing physical protection against pathogen attack.

The unique properties of natural biocomposites have prompted manyscientists to produce composites of cellulose and synthetic polymermatrixes. For example, Favier et al, [Polymer engineering and science37(10): 1732-1739] produced cellulose-latex composites resulting inincreased shear modulus by more than three orders of magnitude of thelatex rubbery state. Such biocomposites have been produced for theautomotive industries and for production of biodegradable plastics.

The use of cellulose binding domains (CBD) for cellulose fibermodification is a well established technology [Shoseyov et al, MicrobiolMol Biol Rev. 70(2):283-95]. Recently, CBD was used for production ofnovel cellulose-protein composite materials when recombinant CBD or CBDdimers, CBD-CBD fusion proteins (CCP), were bound to paper resulting inimproved mechanical and water repelling properties [Levy et al.,Cellulose 9: 91-98]. Furthermore, a recombinant CBD-starch bindingdomain (CSCP) demonstrated cross-bridging ability in different modelsystems composed of insoluble or soluble starch and cellulose [Levy etal., Cellulose 9: 91-98].

In addition to polysaccharide research, biopolymer research has focusedin recent years on fibrous proteins due to their unique mechanicalproperties. These proteins are distinguished by their repetitive aminoacid sequences that confer mechanical strength or flexibility. Amongthese proteins are mammalian collagen and elastin and the arthropodproteins, silkworm silk (Bombyx morii), spider dragline silk andresilin. The unique repetitive sequence of each protein confers itsmechanical properties. For instance, spider silk is extremely strongwhile resilin and elastin are extremely elastic and resilient with arubber-like nature.

Resilin is found in specialized cuticle regions in many insects,especially in areas where high resilience and low stiffness arerequired, or as an energy storage system. It is best known for its rolesin insect flight and the remarkable jumping ability of fleas andspittlebugs. The protein was initially identified in 1960 by Weis-Foghwho isolated it from cuticles of locusts and dragonflies and describedit as a rubber-like material.

Resilin displays unique mechanical properties that combine reversibledeformation with very high resilience. It has been reported to be themost highly efficient elastic material known. The elastic efficiency ofthe material is purported to be 97%; only 3% of stored energy is lost asheat (U.S. Patent Application 20070099231). Resilin shares similarmechanical properties with elastin which is produced in connectivetissues of vertebrates. In humans, elastin is usually found at siteswhere elasticity is required, such as the skin and cartilage (often inassociation with collagen). Elastin-collagen composites also serve as amajor component in arterial walls where it allows the blood vessels tosmooth the pulsatile flow of blood from the heart into a continuous andsteady flow.

In spite of their functional analogy, the sequence homology betweenresilin and elastin is very low, apart from the high abundance ofglycine in both proteins. Nevertheless, the elasticity of both proteinsresults from their architecture of randomly coiled, crosslinkedpolypeptide chains. Resilin is synthesized in the insect cytoplasm andsubsequently secreted to the cuticle where peroxidase enzymes catalyzeits polymerization via formation of di/tri tyrosine bridges, resultingin assembly of a natural protein-carbohydrate composite material withcuticular chitin. Two Drosophila melanogaster Resilin mRNA variants havebeen identified—CG15920-RA and CG15920-RB which differ in the truncationof their chitin binding domains (see FIG. 1A). The major components thatwere annotated are the 17-amino acid long elastic repeats and the 35amino acid-long chitin binding domain of type R&R.

Recently, Elvin et al., 2005, [Nature. 437: 999-1002] successfullyexpressed and polymerized a synthetic, truncated resilin-like gene in E.coli. The synthetic gene consists of the 17 repeats of the native gene.The protein, once expressed, undergoes photochemical crosslinking whichcasts it into a rubber-like biomaterial. U.S. Patent Application20070099231 discloses hybrid resilins comprising resilin and structuralpolypeptides.

Silk proteins are produced by a variety of insects and arachnids, thelatter of which form the strongest silk polymers on earth. The spiderspins as many as seven different kinds of silks, each one beingoptimized to its specific biological function in nature. Dragline silk,used as the safety line and as the frame thread of the spider's web, isan impressive material with a combination of tensile strength andelasticity. Its extraordinary properties are derived from itscomposition as a semicrystalline polymer, comprising crystalline regionsembedded in a less organized “amorphous” matrix. The crystalline regionsconsist of antiparallel β-pleated sheets of polyalanine stretches thatgive strength to the thread, while the predominant secondary structureof the amorphous matrix is the glycine-rich helix which provideselasticity. Most dragline silks consist of at least two differentproteins with molecular masses of up to several hundred kDa. On thebasis of sequence similarities, dragline silk proteins have been groupedinto spidroin1-like (MaSp1) and spidroin2-like (MaSp2) proteins.

As opposed to silkworm silk, isolation of silk from spiders is notindustrially feasible. Spiders produce silk in small quantities, andtheir territorial behavior prevents large amounts thereof from beingharvested in adjacent quarters. Therefore, production of silk proteinthrough recombinant DNA techniques is preferred. For such purposes,widespread use is made of synthetic genes based on a monomer consensusof the native spidroin sequences. These synthetic genes have beensuccessfully expressed in the methyltropic yeast host, Pichia pastoris,in E. coli and in the tobacco and potato plants [Fahnestock S R., andBedzyk L A Appl Microbiol biotechnol 47:33-39 (1997); Fahnestock S R.,and Bedzyk L A, Appl Microbiol biotechnol 47:23-32 (1997), Sceller J. etal. Nature biotechnology 19:573-577 (2001)]. Through such means,laboratory scale amounts of silk-like protein powders are readilyavailable. The final hurdle on the way to the production of manmadesilks lies in the development of an appropriate spinning technologycapable of converting these powders into high performance fibers. Thetendency of these proteins to aggregate in-vitro, bypassing the proteinfolding process, acts as a significant limitation toward successfullyproducing functional silk. The assembly of the proteins from a liquidcrystalline form into a solid silk string is extremely complex, andduplication of the operational function of spider spinning glandsremains a major challenge.

Several attempts have been reported on the preparation of cellulose-silkfibroin composites which were prepared by molecular blending andregeneration of solubilized cellulose and silkworm silk [Freddi G, etal., (1995), J Appl Polymer Sci 56: 1537-1545; Yang, G, et al., (2000) JMembr Sci 210: 177-153]. Recently, Noishiki et al [Noishiki Y, NishiyamaY, Wada M, Kuga S, Magoshi J. (2002) J Appl Polymer Sci 86: 3425-3429]prepared composite cellulose-silk films from solid cellulose whiskersand regenerated silkworm silk, resulting in notably improved mechanicalstrength, with breaking strength and ultimate strain about five timesthose of the constituent materials alone.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided an isolated polypeptide comprising an amino acidsequence encoding a monomer of a fibrous polypeptide attached to aheterologous polysaccharide binding domain, with the proviso that thepolysaccharide binding domain is not a cellulose binding domain.

According to an aspect of some embodiments of the present inventionthere is provided an isolated polypeptide comprising an amino acidsequence encoding a resilin or spider-silk polypeptide attached to aheterologous polysaccharide binding domain.

According to an aspect of some embodiments of the present inventionthere is provided an isolated polynucleotide comprising a nucleic acidsequence encoding an isolated polypeptide comprising an amino acidsequence encoding a monomer of a fibrous polypeptide attached to aheterologous polysaccharide binding domain, with the proviso that thepolysaccharide binding domain is not a cellulose binding domain.

According to an aspect of some embodiments of the present inventionthere is provided an isolated polynucleotide comprising a nucleic acidsequence encoding an isolated polypeptide comprising an amino acidsequence encoding a resilin or spider-silk polypeptide attached to aheterologous polysaccharide binding domain.

According to an aspect of some embodiments of the present inventionthere is provided a nucleic acid construct, comprising a nucleic acidsequence encoding resilin and a cis-acting regulatory element capable ofdirecting an expression of the resilin in a plant.

According to an aspect of some embodiments of the present inventionthere is provided a nucleic acid construct, comprising a nucleic acidsequence encoding spider silk and a cis-acting regulatory elementcapable of directing an expression of the spider silk in a plant.

According to an aspect of some embodiments of the present inventionthere is provided a nucleic acid construct, comprising the isolatedpolynucleotides of the present invention.

According to an aspect of some embodiments of the present inventionthere is provided a cell comprising the nucleic acid constructs of thepresent invention.

According to an aspect of some embodiments of the present inventionthere is provided a plant cell comprising the nucleic acid constructs ofthe present invention.

According to an aspect of some embodiments of the present inventionthere is provided an isolated composite comprising a fibrous polypeptideand a polysaccharide, the fibrous polypeptide being resilin or spidersilk.

According to an aspect of some embodiments of the present inventionthere is provided an isolated composite comprising a fibrous polypeptideand a polysaccharide, the fibrous polypeptide comprising a heterologouspolysaccharide binding domain, the composite being non-immobilized.

According to an aspect of some embodiments of the present inventionthere is provided an isolated composite comprising at least twonon-identical fibrous polypeptides, wherein a first fibrous polypeptideof the at least two non-identical fibrous polypeptide is an isolatedpolypeptide comprising an amino acid sequence encoding a monomer of afibrous polypeptide attached to a heterologous polysaccharide bindingdomain, with the proviso that the polysaccharide binding domain is not acellulose binding domain.

According to an aspect of some embodiments of the present inventionthere is provided an isolated composite comprising at least twonon-identical fibrous polypeptides, wherein a first fibrous polypeptideof the at least two non-identical fibrous polypeptide is an isolatedpolypeptide comprising an amino acid sequence encoding a resilin orspider-silk polypeptide attached to a heterologous polysaccharidebinding domain.

According to an aspect of some embodiments of the present inventionthere is provided a method of generating the isolated composites of thepresent invention, the method comprising contacting the fibrouspolypeptide with the polysaccharide under conditions which allow bindingbetween the fibrous polypeptide and the polysaccharide to generate theisolated composites of the present invention.

According to an aspect of some embodiments of the present inventionthere is provided a use of the isolated composite of the presentinvention for the manufacture of a medicament for the treatment of acartilage or bone disease or condition.

According to an aspect of some embodiments of the present inventionthere is provided a use of the isolated composite of the presentinvention for the manufacture of a medicament for the treatment ofurinary incontinence.

According to an aspect of some embodiments of the present inventionthere is provided a scaffold comprising the isolated composite of thepresent invention.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating a cartilage or bone disease orcondition, the method comprising administering to a subject in needthereof a therapeutically effective amount of the isolated composite ofthe present invention, thereby treating the cartilage disease orcondition.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating urinary incontinence, the methodcomprising administering to a subject in need thereof a therapeuticallyeffective amount of the isolated composite of the present invention,thereby treating urinary incontinence.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising the isolatedcomposite of the present invention.

According to an aspect of some embodiments of the present inventionthere is provided a cosmetic composition comprising the isolatedcomposite of the present invention.

According to some embodiments of the invention, the fibrous polypeptideis selected from the group consisting of resilin, elastin, spider silk,silk-worm silk, collagen and mussel byssus protein.

According to some embodiments of the invention, the fibrous polypeptidecomprises resilin.

According to some embodiments of the invention, the fibrous polypeptidecomprises spider silk.

According to some embodiments of the invention, the resilin comprises anamino acid sequence as set forth in SEQ ID NO: 8.

According to some embodiments of the invention, the resilin comprises anamino acid sequence as set forth in SEQ ID NO: 9

According to some embodiments of the invention, the polypeptide furthercomprises an amino acid sequence as set forth in SEQ ID NOs: 52 or 53.

According to some embodiments of the invention, the polysaccharidebinding domain is selected from the group consisting of a chitin bindingdomain, a starch binding domain, a dextran binding domain, a glucanbinding domain, a chitosan binding domain, an alginate binding domainand an hyaluronic acid binding domain.

According to some embodiments of the invention, the polysaccharidebinding domain is selected from the group consisting of a chitin bindingdomain, a cellulose binding domain, a starch binding domain, a dextranbinding domain, a glucan binding domain, a chitosan binding domain, analginate binding domain and an hyaluronic acid binding domain.

According to some embodiments of the invention, the isolated polypeptideis as set forth in SEQ ID NOs: 11-13 and SEQ ID NOs. 32-36.

According to some embodiments of the invention, the spider silkcomprises an amino acid sequence as set forth in SEQ ID NO: 16 or SEQ IDNO: 26.

According to some embodiments of the invention, the polynucleotidecomprises a nucleic acid sequence selected from the group consisting ofSEQ ID NO: 17-22, 24, 28 and 29.

According to some embodiments of the invention, the nucleic acidconstruct further comprises at least one cis-acting regulatory element.

According to some embodiments of the invention, the cis-actingregulatory element is a plant promoter.

According to some embodiments of the invention, the plant promoter is arbcS1 promoter.

According to some embodiments of the invention, the nucleic acidconstruct further comprises a nucleic acid sequence encoding a vacuolarsignal sequence.

According to some embodiments of the invention, the cis-actingregulatory sequence is a terminator sequence.

According to some embodiments of the invention, the terminator sequenceis a rbcS1 sequence.

According to some embodiments of the invention, the cell is a plantcell.

According to some embodiments of the invention, the polysaccharide isselected from the group consisting of chitin, cellulose, starch,dextran, glucan, chitosan, alginate and hyaluronic acid.

According to some embodiments of the invention, the fibrous polypeptidecomprises a polysaccharide binding domain.

According to some embodiments of the invention, the polysaccharidebinding domain is a heterologous polysaccharide binding domain.

According to some embodiments of the invention, the polysaccharidebinding domain comprises a chitin binding domain, a cellulose bindingdomain, a chitosan binding domain, an alginate binding domain, a starchbinding domain, a dextran binding domain, a glucan binding domain and anhyaluronic acid binding domain.

According to some embodiments of the invention, the fibrous polypeptideis selected from the group consisting of mussel byssus protein, resilin,silkworm silk protein, spider silk protein, collagen, elastin orfragments thereof.

According to some embodiments of the invention, the isolated compositefurther comprises an additional fibrous polypeptide, wherein theadditional fibrous polypeptide is different to the fibrous polypeptide,the additional fibrous polypeptide being selected from the groupconsisting of mussel byssus protein, resilin, silkworm silk protein,spider silk protein, collagen, elastin and fragments thereof.

According to some embodiments of the invention, the isolated compositeis crosslinked.

According to some embodiments of the invention, the isolated compositeis non-crosslinked.

According to some embodiments of the invention, the method furthercomprises crosslinking the composite following the contacting.

According to some embodiments of the invention, the crosslinking isaffected by a method selected from the group consisting of photochemicalcrosslinking, enzymatic crosslinking, chemical crosslinking and physicalcrosslinking.

According to some embodiments of the invention, the method furthercomprises coating the composite with an additional fibrous polypeptide,the coating being effected following the crosslinking the composite.

According to some embodiments of the invention, the method furthercomprises binding the fibrous polypeptide with an additional fibrouspolypeptide prior to the contacting.

According to some embodiments of the invention, the additional fibrouspolypeptide is selected from the group consisting of a mussel byssusprotein, spider silk protein, collagen, elastin, and fibronectin, andfragments thereof.

According to some embodiments of the invention, the polysaccharide isselected from the group consisting of a chitin, a cellulose, a starch, adextran, a glucan, a chitosan, an alginate, a carboxymethyl celluloseand an hyaluronic acid.

According to some embodiments of the invention, the use is for cartilagerepair, knee repair, meniscus repair a knee lubricant and disc repair.

According to some embodiments of the invention, the administering iseffected locally.

According to some embodiments of the invention, the locallyadministering is effected by intra-articular administration.

According to some embodiments of the invention, the intra-articularadministration comprises administration into a joint selected from thegroup consisting of a knee, an elbow, a hip, a sternoclavicular, atemporomandibular, a carpal, a tarsal, a wrist, an ankle, anintervertebral disk and a ligamentum flavum.

According to some embodiments of the invention, the cartilage disease orcondition is selected from the group consisting of osteoarthritis,limited joint mobility, gout, rheumatoid arthritis, osteoarthritis,chondrolysis, scleroderma, degenerative disc disorder and systemic lupuserythematosus.

According to some embodiments of the invention, the administering iseffected by injection into an area surrounding the urethra.

According to some embodiments of the invention, the composition isformulated as a gel, a strip, an injectable, or a foam.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIGS. 1A-B is a schematic illustration (FIG. 1A) and scan (FIG. 1B)illustrating the size and structure of the resilin gene from D.melonogaster. FIG. 1A illustrates the schematic structure of Drosophilamelanogaster resilin variant A CG15920-RA gene; S.P.; cuticular signalpeptide, ChBD R&R; chitin binding domain type R&R. Variant B CG15920-RBcontains a truncated chitin binding domain. FIG. 1B illustrates RT-PCRresults of amplification of the resilin gene from D. melonogaster. Theresilin cDNA is highlighted by the red rectangle. The thick band wasformed due to the presence of two resilin variants. The band in thecontrol reaction lanes (-RT) indicates the genomic gene that containsone intron and therefore migrates slower than the RT-PCR product.

FIG. 2 is a scheme of the multiple cloning site of the pHis-parallel3expression vector (SEQ ID NO: 54).

FIGS. 3A-B are scans of PCR results of CBD-Resilin (SEQ ID NO: 18)construction. FIG. 3A illustrates the first round PCR of the separatereactions of CBD (left) and resilin (right) sequence amplification. TheCBD sequence contains a resilin-matching overhang on the 3′ prime whilethe Resilin contains CBD-matching overhang on the 5′ prime. FIG. 3Billustrates the PCR result of the second round following mixture of 1 lof both products from round one. Note the increased molecular weight ofthe linked sequences.

FIG. 4 is a scan of a Coomassie blue-stained SDS PAGE analysis of asmall scale batch purification of 6H-Res-ChBD (SEQ ID NO: 55) expressedin bacteria. S: soluble protein fraction of the lyzed cells; IB:inclusion bodies; UB: unbound fraction removed by centrifugation; W:wash; E1,E2: eluted protein with 0.4M imidazole. MW: protein molecularweight marker.

FIG. 5 is a scan of a Coomassie blue-stained SDS PAGE analysisillustrating the results of a cellulose and chitin binding assay withthe affinity-purified 6H-Res-ChBD protein (SEQ ID NO: 55). T: Proteinpulled down by HIS-Select® affinity product; UB: unbound fractionremoved by centrifugation; W: wash fraction; B: bound fraction elutedfrom cellulose/chitin pellets by boiling with SDS-PAGE sampleapplication buffer. MW: molecular weight marker.

FIGS. 6A-C are scans of Coomassie blue-stained SDS PAGE analysesillustrating the results of a cellulose and chitin binding assay of acrude extract comprising 6H-Res-ChBD (SEQ ID NO: 55). T: crude lysate;W: wash fraction UB; unbound fraction removed by centrifugation; B:bound fraction eluted from cellulose/chitin pellets by boiling withSDS-PAGE sample application buffer. B 1:5: bound fraction diluted fivetime to the true initial load concentration. MW: protein molecularweight marker.

FIG. 7 is a photograph of an SDS PAGE analysis illustrating the resultsof a Res-ChBD (SEQ ID NO: 55) heat stability assay. UH: unheatedprotein. Lanes 2-4: samples subjected to 85° C. for 15, 30, 60 minutes,respectively. MW: protein molecular weight marker.

FIGS. 8A-B illustrate a small scale affinity purification of6H-Res-ChBD. FIG. 8A: Chromatogram illustrating purification of Res-ChBDon a Ni-NTA column. The observed peak was eluted with 220 mM imidazoleat min 13.4. FIG. 8B: SDS-PAGE analysis of small scale Ni-NTApurification of 6H-Res-ChBD (SEQ ID NO: 55). 6-17: number of FPLCfractions loaded on the gel; FT: column flow through. Fractions 9-18were collected for further analysis.

FIG. 9 is a scan of an SDS-PAGE analysis of photochemical polymerizationof 6H-Res-ChBD (SEQ ID NO: 55). UH: unheated affinity purified6H-Res-ChBD; H: purified 6H-Res-ChBD incubated at 85° C. for 15 minutes;P: 6H-Res-ChBD treated with Ru(bpy)3Cl2.6; H2O and ammonium persulfateprior to subjection to sunlight. The treatment resulted in highmolecular weight products that could not get into the gel and remainedin the loading wells (indicated by arrow).

FIGS. 10A-B illustrate a medium scale affinity purification of6H-Res-ChBD (SEQ ID NO: 55). FIG. 10A: Chromatogram of 6H-Res-ChBDpurification on a Ni-NTA column. The protein peak observed was elutedwith 180 mM imidazole at min 22.7. FIG. 10B: Coomassie blue stainedSDS-PAGE analysis of a medium scale Ni-NTA purification of 6H-Res-ChBD.1-11: numbers of FPLC fractions loaded on the gel; FT: column flowthrough; W: column wash.

FIG. 11 is a scan of a Coomassie-blue stained SDS-PAGE analysis of aNi-NTA-purified recombinant resilin (SEQ ID NO: 56). Lanes 1-8: FPLCfractions; FT: column flow through. Fractions 4-7 correspond to thepurified resilin.

FIG. 12 is a scan of a Coomassie blue stained SDS-PAGE analysis ofCBD-resilin (SEQ ID NO: 57), marked by the arrow, post lysis of thebacteria. The protein was detected almost exclusively in the inclusionbodies (IB).

FIG. 13 is a scan of a Coomassie blue stained SDS-PAGE analysis of thecellulose binding capacity of affinity purified CBD-resilin (SEQ ID NO:57). T: Ni-NTA purified CBD-resilin; UB: unbound fraction removed bycentrifugation; W: wash fraction; B: bound fraction eluted fromcellulose/chitin pellets by boiling with SDS-PAGE sample applicationbuffer.

FIG. 14 is a scan of a Coomassie blue stained SDS-PAGE analysis ofCBD-resilin refolded via the Aktaprime™ Plus FPLC automated refoldingsystem (SEQ ID NO: 57) bound to cellulose. T; Ni-NTA purifiedCBD-resilin; B: bound fraction eluted from cellulose pellets by boilingwith SDS-PAGE sample application buffer; UB: unbound fraction removed bycentrifugation.

FIG. 15 is a model of a composite of cellulose and spider silk.

FIG. 16 is a scan of a Coomassie-stained SDS-PAGE analysis of Ni-NTApurified recombinant resilin-CBD (SEQ ID NO: 58). Samples 9-17 were theFPLC-ÄKTAprime™ plus fractions. Fractions 15-16 correspond to theresilin-CBD peak as observed at O.D. 280 nm.

FIG. 17 is a scan of a Coomassie-stained SDS-PAGE analysis ofresilin-CBD (SEQ ID NO: 58) following heat treatment and a cellulosebinding assay. UH: Unheated protein; H: Protein incubated at 85° C. for15 minutes; T: Total protein (affinity chromatography product); B: Boundfraction eluted by boiling the cellulose pellet with ×2 SAB; UB: Unboundfraction removed by centrifugation.

FIG. 18 is a scan of a Coomassie-stained SDS-PAGE analysis of thesolubility of resilin (SEQ ID NO: 56) and resilin-ChBD (SEQ ID NO: 55)proteins under different pH conditions, following gradual titration with2M HCl.

FIG. 19 is a scan of a Coomassie-stained SDS-PAGE analysis of resilinsamples that were subjected to light-induced polymerization underdifferent pH conditions in the presence (+) or absence (−) ofRu(bpy)3Cl2.6; H2O and APS. Control samples of Res-ChBD (SEQ ID NO: 55)proteins (pH 7.4) were subjected to similar crosslinking conditions. Thearrow points out the high molecular weight products in samplescontaining the crosslinkers.

FIG. 20 is a scan of a Coomassie-stained SDS-PAGE analysis of resilinpolymerized by the MCO method in either a phosphate buffer orwater-based reaction solution. A high molecular weight product wasformed both in phosphate buffer and H2O. The reaction carried out in H2Odemonstrated a polymerization effect in the reaction with H2O2 only.

FIGS. 21A-B are photographs illustrating the generation of a compositeof the present invention. FIG. 21A-Opening of the Teflon mold followingphotochemical crosslinking of the 6H-Res-ChBD-cellulose composites. FIG.21B—left and middle are resulting composite polymers of 150 and 75 μlsamples of 6H-Res-ChBD-cellulose whiskers, respectively, while thesample on the right is that received from the 150 μl sample of pure6H-Res-ChBD polymer casted in the absence of cellulose whiskers.

FIGS. 22A-B are scans illustrating SDS PAGE analyses of E. Coli proteinsfollowing overexpression of an exemplary spider silk of the presentinvention. FIG. 22A—Coomassie blue-stained SDS-PAGE analysis of total E.coli proteins. Proteins were stained with Coomassie blue. Lane 1—proteinmolecular weight marker, lane 2—control bacteria transformed with emptyvector, lane 3—proteins collected from SpS (SEQ ID NO: 33)-expressingbacteria, lane 4—proteins of SpS-CBD-expressing bacteria (SEQ ID NO:34). FIG. 22B—and Instant blue-stained SDS-PAGE analysis of soluble (S)and insoluble (IB) E. coli proteins. Proteins were stained withCoomassie blue. Lane 1—protein molecular weight marker, lanes2-3—proteins of SpS (SEQ ID NO: 33)-expressing bacteria, S and IB,respectively. Lanes 4-5—proteins of SpS-CBD (SEQ ID NO: 34)-expressingbacteria, S and IB, respectively.

FIGS. 23A-B are scans illustrating SDS-PAGE analyses of FPLC-purified6H-SpS (SEQ ID NO: 33) and 6H-SpS-CBD (SEQ ID NO: 34) expressed in E.Coli. FIG. 23A—SDS-PAGE analysis of FPLC fractions of Ni-NTA-purifiedSpS proteins. Lane 1—protein molecular weight marker, lanes 2-4—solubleproteins of empty vector-transformed E. Coli control lysates, SpS (SEQID NO: 33) and SpS-CBD (SEQ ID NO: 34) samples, respectively, prior toNi-NTA purification. Lanes 4-7—purified protein fractions of control,SpS (SEQ ID NO: 33), SpS-CBD (SEQ ID NO: 34), respectively, followingNi-NTA purification. FIG. 23B—Western blot analysis of the same samplesas described in FIG. 17A with anti-6His antibody.

FIGS. 24A-C are graphs illustrating FPLC purification of 6H-SpS (SEQ IDNO: 33) and 6H-SpS-CBD (SEQ ID NO: 34). FIG. 24A—Chromatogram of thepurification of control E. coli proteins on a Ni-NTA column. FIG.24B—Chromatogram of the purification of 6H-SpS (SEQ ID NO: 33), onNi-NTA column. FIG. 24C-Chromatogram of the purification of 6H-SpS-CBD(SEQ ID NO: 34), on a Ni-NTA column.

FIG. 25 is a scan of an SDS-PAGE analysis of a qualitative cellulosebinding assay of affinity-purified SpS (SEQ ID NO: 33) and SpS-CBD (SEQID NO: 34). Lane 1—protein molecular weight marker, Lanes 2-4—spidersilk cellulose binding assay: lane 2—SpS after Ni-NTA purification, lane3—cellulose-bound protein, lane 4—unbound protein. The unbound proteinis diluted 1:10 in comparison to protein concentration in lane 2. Lanes5-7—SpS-CBD cellulose binding assay: lane 5—SpS-CBD after Ni-NTApurification, lane 6—cellulose-bound protein, lane 7—unbound protein.The unbound protein is diluted 1:10 in comparison to proteinconcentration in lane 5.

FIG. 26 is a graph of an adsorption/desorption isotherm. CBDClostridiumcellulovorans (CBDclos) (SEQ ID NO: 10), SpS (SEQ ID NO: 33) and SpS-CBD(SEQ ID NO: 34), at different concentrations, were allowed to adsorb tocellulose to the point of equilibrium (B). After equilibrium wasreached, the highest protein concentration containing mixture wasdiluted to allow desorption (R).

FIGS. 27A-B are scans of Western blot analyses of lysates of CBD-SpS12(SEQ ID NO:35) and SpS6-CBD-SpS6 (SEQ ID NO:36)-expressing plants, usinganti-CBD antibody for immunodetection. FIG. 27A—Tobacco plantsexpressing and accumulating CBD-SpS12 (SEQ ID NO:35) in the apoplast.Lane 1—protein molecular weight marker, lane 2—wild type tobacco plantlysates, Lanes 3-8—lysates of transgenic tobacco plant numbers13.1-13.6, respectively. S—soluble proteins, P—insoluble proteins. FIG.27B—Tobacco plants expressing SpS6-CBD-SpS6 (SEQ ID NO:36) in thecytoplasm. Lane 1—protein molecular weight marker, lane 2—wild typetobacco plant lysates, Lanes 3-8—Lysates of transgenic tobacco plantnumbers 6.1-6.6, respectively. S—soluble proteins, P—insoluble proteins.

FIGS. 28A-B are scans of Western blot analyses of the SpS6-CBD-SpS6 (SEQID NO: 36) purification procedure, using anti-CBD antibody forimmunodetection. FIG. 28A—Lane 1—protein molecular weight marker, lane2—soluble proteins of wild type tobacco plant extracts, Lane 3—insolubleproteins of wild type tobacco, lane 4—soluble proteins of transgenictobacco plant #6.4, lane 5—insoluble proteins of transgenic tobaccoplant #6.4, lane 6—soluble proteins eluted from the insoluble fractionof #6.4 transgenic tobacco plant SpS6-CBD-SpS6 (SEQ ID NO: 36), lane7—insoluble proteins eluted from the insoluble fraction of 6.4transgenic tobacco plant SpS6-CBD-SpS6. FIG. 28B illustrates the heatstability and pH solubility of SpS6-CBD-SpS6 (SEQ ID NO: 36). Lane1—protein molecular weight marker, lane 2—soluble proteins eluted fromthe insoluble fraction of the plant extract (as shown in FIG. 24A lane6), lanes 3-6—heat stability assay at 60, 70, 80 and 90° C.respectively. Lanes 7-12—pH solubility test under pH=8, 7, 6, 5, 4, 3,respectively.

FIG. 29 is a Coomassie-stained SDS-PAGE analysis of metal-catalyzedpolymerization of silk. Lane 1—protein molecular weight marker; lanes2-5—reaction analysis of SpS (SEQ ID NO: 33) dialyzed against DDW: lane2—protein solution without H2O2 or CuCl2, lane 3—polymerization reactionincluding H2O2 and CuCl2, lane 4—protein solution with addition of H2O2only, lane 5—protein solution with the addition of CuCl2 only. Lanes6-9: reaction analysis of SpS dialyzed against 50 mM sodium phosphate(pH 7.5): lane 6—protein solution without H2O2 or CuCl2, lane7—polymerization reaction including H2O2 and CuCl2, lane 8—proteinsolution with addition of H2O2 only, lane 9—protein solution with theaddition of CuCl2 only.

FIG. 30 is a Coomassie-stained SDS-PAGE analysis of SpS spongepreparation. Lane 1—protein molecular weight marker, lane 2—solubleprotein before sponge preparation procedure, lane 3—soluble proteinafter dialysis against 50 mM sodium phosphate (pH 7.5), lane 4—solubleprotein after dialysis against DDW, lane 5—soluble protein afterconcentration to ˜50 mg/ml. The sample was diluted ×50 in order toconfirm that there was no protein loss.

FIGS. 31A-C depict the results of DSC analysis of SpS-cellulose whiskersponges. A—DSC thermogram analysis of cellulose whiskers sponge; B—DSCthermogram analysis of SpS sponge; C—DSC thermogram analysis of 70%whiskers/30% SpS sponge.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates tocompositions comprising fibrous polypeptides and polysaccharides anduses of same. The fibrous polypeptides may comprise an endogenouspolysaccharide binding domain or a heterologous polysaccharide bindingdomain.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

In a search to identify novel composite biomaterials with superiormechanical properties for use in medical, industrial and otherapplications, the present inventors have generated novel fibrouspolypeptides that enable directional binding and polymerization onpolysaccharides.

Whilst reducing the present invention to practice, the present inventorsgenerated and purified both resilin and spider-silk fusion proteins.Exemplary fusion proteins generated include resilin-chitin bindingdomain (Res-ChBD) (FIGS. 4-10, 18 and 19); resilin-cellulose bindingdomain (Res-CBD) (FIGS. 12-14, 16-17) and; spider-silk-cellulose bindingdomain (FIGS. 23-28).

Thus, according to one aspect of the present invention, there isprovided an isolated polypeptide comprising an amino acid sequenceencoding a monomer of a fibrous polypeptide attached to a heterologouspolysaccharide binding domain.

As used herein, the phrase “fibrous polypeptide” refers to a polypeptidethat consists of a plurality of monomer chains arranged in a matrix soas to form fibers or sheets. Fibrous proteins are described in D. Voet &J. G. Voet, “Biochemistry” (2d ed., John Wiley & Sons, New York, 1995,pp. 153-162), incorporated herein by this reference.

Examples of fibrous polypeptides include, but are not limited to,resilin, elastin, spider silk, silk-worm silk, collagen and musselbyssus protein.

As used herein, the term “resilin” refers to an elastic polypeptide,capable of forming a fiber, wherein each monomer thereof comprises atleast two repeating units of the sequence as set forth in SEQ ID NO: 45.According to one embodiment, the repeating unit comprises a sequence asset forth in SEQ ID NO: 8. GenBank Accession Nos. of non-limitingexamples of resilin are listed in Table 1 below. A resilin of thepresent invention also refers to homologs (e.g. polypeptides which areat least 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 87%, at least 89%, atleast 91%, at least 93%, at least 95% or more say 100% homologous toresilin sequences listed in Table 1 as determined using BlastP softwareof the National Center of Biotechnology Information (NCBI) using defaultparameters). The homolog may also refer to a deletion, insertion, orsubstitution variant, including an amino acid substitution, thereof andbiologically active polypeptide fragments thereof.

Table 1 below lists examples of resilin NCBI sequence numbers.

TABLE 1 Exemplary resilin NCBI sequence number Organism NP 995860Drosophila melanogaster NP 611157 Drosophila melanogaster Q9V7U0Drosophila melanogaster AAS64829 Drosophila melanogaster AAF57953Drosophila melanogaster XP 001817028 Tribolium castaneum XP001947408Acyrthosiphon pisum

According to one embodiment, the polypeptide sequence of resilin is setforth in SEQ ID NO: 9.

As used herein, the term “elastin” refers to an elastic polypeptide,capable of forming a fiber, wherein each monomer thereof comprises atleast two repeating units of the sequence as set forth in SEQ ID NO: 46.GenBank Accession Nos. of non-limiting examples of elastin are listed inTable 2 below. An elastin of the present invention also refers tohomologs (e.g., polypeptides which are at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 87%, at least 89%, at least 91%, at least 93%, atleast 95% or more say 100% homologous to elastin sequences listed inTable 2 as determined using BlastP software of the National Center ofBiotechnology Information (NCBI) using default parameters). The homologmay also refer to a deletion, insertion, or substitution variant,including an amino acid substitution, thereof and biologically activepolypeptide fragments thereof.

Table 2 below lists examples of elastin NCBI sequence numbers.

TABLE 2 Exemplary elastin Organism NCBI sequence number Bos taurusNP786966 mouse NP 031951 rat NP 036854 Human AAC98395 sheep I47076

As used herein, the term “spider silk” refers to a polypeptide capableof forming a fiber which is comprised of spider silk, wherein eachmonomer thereof comprises at least two repeating units of the sequenceset forth in SEQ ID NO: 26. According to one embodiment, the polypeptidechain comprises a spidroin 1 amino acid sequence. According to anotherembodiment, the polypeptide chain comprises a spidroin 2 amino acidsequence. According to one embodiment, the spider silk is draglinespider silk. GenBank Accession Nos. of non-limiting examples ofspidroins 1 and 2 are listed in Table 3 below. A spider silk polypeptideof the present invention also refers to homologs (e.g., polypeptideswhich are at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 87%, atleast 89%, at least 91%, at least 93%, at least 95% or more say 100%homologous to spider silk sequences listed in Table 3 as determinedusing BlastP software of the National Center of BiotechnologyInformation (NCBI) using default parameters). The homolog may also referto a deletion, insertion, or substitution variant, including an aminoacid substitution, thereof and biologically active polypeptide fragmentsthereof.

Table 3 below lists examples of spider silk NCBI sequence numbers.

TABLE 3 Exemplary spider silk NCBI sequence Spider silk polypeptidenumber Spidroin 1 P19837 Spidroin 1 AAC38957 Spidroin 2 ABR68858Spidroin 2 AAT75317 Spidroin 2 P46804

According to one embodiment, the polypeptide sequence of the spider silkpolypeptide is set forth in SEQ ID NO: 16 or SEQ ID NO: 38.

As used herein, the term “silkworm silk” refers to a silk polypeptidederived from silkworm, capable of forming a fiber. GenBank AccessionNos. of non-limiting examples of silkworm silk polypeptides are listedin Table 4 below. A silkworm silk polypeptide of the present inventionalso refers to homologs (e.g., polypeptides which are at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 87%, at least 89%, at least 91%, atleast 93%, at least 95% or more say 100% homologous to silkworm silksequences listed in Table 4 as determined using BlastP software of theNational Center of Biotechnology Information (NCBI) using defaultparameters). The homolog may also refer to a deletion, insertion, orsubstitution variant, including an amino acid substitution, thereof andbiologically active polypeptide fragments thereof.

Table 4 below lists examples of silkworm silk NCBI sequence numbers.

TABLE 4 Exemplary silkworm silk NCBI sequence number AAL83649 AAA27839NP 001106733 NP001037488 Caa35180

As used herein, the term “collagen” refers to an assembled collagentrimer, which in the case of type I collagen includes two alpha 1 chainsand one alpha 2 chain. A collagen fiber is collagen which is devoid ofterminal propeptides C and N. Contemplated collagens include types I,II, III, V, XI, and biologically active fragments therefrom. Thecollagen may be comprised of procollagen, atelocollagen or telocollagen.A collagen of the present invention also refers to homologs (e.g.,polypeptides which are at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 87%, at least 89%, at least 91%, at least 93%, at least 95% ormore say 100% homologous to collagen sequences listed in Table 1 asdetermined using BlastP software of the National Center of BiotechnologyInformation (NCBI) using default parameters). The homolog may also referto a deletion, insertion, or substitution variant, including an aminoacid substitution, thereof and biologically active polypeptide fragmentsthereof.

Table 5 below lists examples of collagen NCBI sequence numbers.

TABLE 5 Exemplary human collagen NCBI sequence number P02452 P08123

As used herein, the phrase “mussel byssus protein” refers to thepolypeptide found in the byssal threads of mussels comprising bothcollagen and elastin domains (e.g. Col-P or Col-D). A mussel byssusprotein of the present invention also refers to homologs (e.g.,polypeptides which are at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 87%, at least 89%, at least 91%, at least 93%, at least 95% ormore say 100% homologous to mussel byssus sequences as set forth in NCBIsequence numbes AAB34042 and as determined using BlastP software of theNational Center of Biotechnology Information (NCBI) using defaultparameters).

The homolog may also refer to a deletion, insertion, or substitutionvariant, including an amino acid substitution, thereof and biologicallyactive polypeptide fragments thereof.

As mentioned, the isolated polypeptides of the present inventioncomprise a monomer of a fibrous polypeptide attached to a heterologouspolysaccharide binding domain.

As used herein, the qualifier “heterologous” when relating to theheterologous polysaccharide binding domains of the fibrous polypeptidesof the present invention indicates that the heterologous polysaccharidebinding domain is not naturally found in that fibrous polypeptide towhich it is fused.

The phrase “polysaccharide binding domain” refers to an amino acidsequence which binds a polysaccharide with a minimal dissociationconstant (Kd) of about 10 M [Tomme P, Boraston A, McLean B, Kormos J,Creagh A L, Sturch K, Gilkes N R, Haynes C A, Warren R A, Kilburn D G(1998) Characterization and affinity applications of cellulose-bindingdomains. J Chromatogr B Biomed Sci Appl. 715(1):283-96, Boraston A B,Bolam D N, Gilbert H J, Davies G J. (2004) Carbohydrate-binding modules:fine-tuning polysaccharide recognition. Biochem J. 382(Pt 3):769-81].Typically, the polysaccharide binding domain comprises at least afunctional portion of a polysaccharide binding domain of apolysaccharidase or a polysaccharide binding protein.

It will be appreciated that the fibrous polypeptide may be joineddirectly to the polysaccharide binding domain or may be joined via alinker. Amino acid sequences of exemplary linkers contemplated for thepresent invention are set forth in SEQ ID NOs: 52 and 53.

Exemplary polysaccharide binding domains include but are not limited toa chitin binding domain (examples of which are set forth in SEQ ID NO:39 and 40), a starch binding domain (an example of which is set forth inSEQ ID NO: 41), a dextran binding domain (an example of which is setforth in SEQ ID NO: 42), a glucan binding domain, a chitosan bindingdomain (see for example Chen, H P; Xu, L L, (2005) J. of IntegrativePlant Biology 47(4): 452-456), an alginate binding domain (an example ofwhich is set forth in SEQ ID NO: 43) and an hyaluronic acid bindingdomain (an example of which is set forth in SEQ ID NO: 44).

According to this aspect of the present invention, when the fibrouspolypeptide comprises resilin or spider silk, the polysaccharide bindingdomain may also be a cellulose binding domain.

Table 6 below lists exemplary sources of polysaccharide binding domainswhich are contemplated for use in the present invention.

TABLE 6 Sources of polysaccharide binding domains Proteins Containingthe Binding Binding Domain Domain Cellulose Binding β-glucanases(avicelases, CMCases, Domains¹ cellodextrinases) exoglucanses orcellobiohydrolases cellulose binding proteins xylanases mixedxylanases/glucanases esterases chitinases β-1,3-glucanasesβ-1,3-(β-1,4)-glucanases (β-)mannanases β-glucosidases/galactosidasescellulose synthases (unconfirmed) Starch/Maltodextrin α-amylases^(2,3)Binding Domains β-amylases^(4,5) pullulanases glucoamylases^(6,7)cyclodextrin glucotransferases⁸⁻¹⁰ (cyclomaltodextringlucanotransferases) maltodextrin binding proteins¹¹ Dextran BindingDomains (Streptococcal) glycosyl transferases¹² dextran sucrases(unconfirmed) Clostridial toxins^(13,14) glucoamylases⁶ dextran bindingproteins β-Glucan Binding Domains β-1,3-glucanases^(15,16)β-1,3-(β-1,4)-glucanases (unconfirmed) β-1,3-glucan binding protein¹⁷Chitin Binding Domains chitinases chitobiases chitin binding proteins(see also cellulose binding domains) Heivein ¹Gilkes et al., Adv.Microbiol Reviews, (1991) 303-315. ²Søgaard et al., J. Biol. Chem.(1993) 268: 22480. ³Weselake et al., Cereal Chem. (1983) 60: 98.⁴Svensson et al., J. (1989) 264: 309. ⁵Jespersen et al., J. (1991) 280:51. ⁶Belshaw et al., Eur. J. Biochem. (1993) 211: 717. ⁷Sigurskjold etal., Eur. J. Biochem. (1994) 225: 133. ⁸Villette et al., Biotechnol.Appl. Biochem. (1992) 16: 57. ⁹Fukada et al., Biosci. Biotechnol.Biochem. (1992) 56: 556. ¹⁰Lawson et al., J. Mol. Biol. (1994) 236: 590.¹⁴von Eichel-Streiber et al., Mol. Gen. Genet. (1992) 233: 260. ¹⁵Kleblet al., J. Bacteriol. (1989) 171: 6259. ¹⁶Watanabe et al., J. Bacteriol.(1992) 174: 186. ¹⁷Duvic et al., J. Biol. Chem. (1990): 9327.

Table 7 below lists an overview of enzymes with chitin binding domainswhich are contemplated for use as the polysaccharide domains of thepresent invention.

TABLE 7 Source (strain) Enzyme Accession No. Ref.¹ Bacterial enzymesType I Aeromonas sp. (No10S-24) Chi D31818 1 Bacillus circulans (WL-12)ChiA1 P20533/M57601/A38368 2 Bacillus circulans (WL-12) ChiDP27050/D10594 3 Janthinobacterium lividum Chi69 U07025 4 Streptomycesgriseus Protease C A53669 5 Type II Aeromonas cavia (K1) Chi U09139 6Alteromonas sp (0-7) Chi85 A40633/P32823/D13762 7 Autographa californica(C6) NPH-128^(a) P41684/L22858 8 Serratia marcescens ChiAA25090/X03657/L01455/P07254 9 Type III Rhizopus oligosporus (IFO8631)Chi1 P29026/A47022/D10157/S27418 10 Rhizopus oligosporus (IFO8631) Chi2P29027/B47022/D10158/S27419 10 Saccharomyces cerevisiae ChiS50371/U17243 11 Saccharomyces cerevisiae Chi1 P29028/M74069 12 (DBY939)Saccharomyces cerevisiae Chi2 P29029/M7407/B41035 12 (DBY918) Plantenzymes Hevein superfamily Allium sativum Chi M94105 13 Amaranthuscaudatus AMP-1^(b) P27275/A40240 14, 15 Amaranthus caudatus AMP-2^(b)S37381/A40240 14, 15 Arabidopsis thaliana ChiB P19171/M38240/B45511 16(cv. colombia) Arabidopsis thaliana PHP^(c) U01880 17 Brassica napus ChiU21848 18 Brassica napus Chi2 Q09023/M95835 19 Hevea brasiliensisHev1^(d) P02877/M36986/A03770/A38288 20, 21 Hordeum vulgare Chi33 L3421122 Lycopersicon esculentum Chi9 Q05538/Z15140/S37344 23 Nicotianatabacum CBP20^(e) S72424 24 Nicotiana tabacum Chi A21091 25 Nicotianatabacum (cv. Havana) Chi A29074/M15173/S20981/S19855 26 Nicotianatabacum (FB7-1) Chi JQ0993/S0828 27 Nicotiana tabacum (cv. Samsun) ChiA16119 28 Nicotiana tabacum (cv. Havana) Chi P08252/X16939/S08627 27Nicotiana tabacum (cv. BY4) Chi P24091/X51599/X64519//S13322 26, 27, 29Nicotiana tabacum (cv. Havana) Chi P29059/X64518/S20982 26 Oryza sativum(IR36) ChiA L37289 30 Oryza sativum ChiB JC2253/S42829/Z29962 31 Oryzasativum Chi S39979/S40414/X56787 32 Oryza sativum (cv. Japonicum) ChiX56063 33 Oryza sativum (cv. Japonicum) Chi1 P24626/X54367/S14948 34Oryza sativum Chi2 P25765/S15997 35 Oryza sativum (cv. Japonicum) Chi3D16223 Oryza sativum ChiA JC2252/S42828 30 Oryza sativum Chi1 D16221 32Oryza sativum (IR58) Chi U02286 36 Oryza sativum Chi X87109 37 Pisumsativum (cv. Birte) Chi P36907/X63899 38 Pisum sativum (cv. Alcan) Chi2L37876 39 Populus trichocarpa Chi S18750/S18751/X59995/P29032 40 Populustrichocarpa (H11-11) Chi U01660 41 Phaseolus vulgaris (cv. Saxa) ChiA24215/S43926/Jq0965/P36361 42 Phaseolus vulgaris (cv. Saxa) ChiP06215/M13968/M19052/A25898 43, 44, 45 Sambucus nigra PR-3^(f) Z46948 46Secale cereale Chi JC2071 47 Solanum tuberosum ChiB1 U02605 48 Solanumtuberosum ChiB2 U02606 48 Solanum tuberosum ChiB3 U02607/S43317 48Solanum tuberosum ChiB4 U02608 48 Solanum tuberosum WIN-1^(g)P09761/X13497/S04926 49 (cv. Maris Piper) Solanum tuberosum WIN-2^(g)P09762/X13497/S04927 49 (cv. Maris Piper) Triticum aestivum ChiS38670/X76041 50 Triticum aestivum WGA-1^(h) P10968/M25536/S09623/S0728951, 52 Triticum aestivum WGA-2^(h) P02876/M25537/S09624 51, 53 Triticumaestivum WGA-3^(h) P10969/J02961/S10045/A28401 54 Ulmus americana(NPS3-487) Chi L22032 55 Urtica dioica AGL^(i) M87302 56 Vignaunguiculata Chi1 X88800 57 (cv. Red caloona) ^(a)NHP: nuclearpolyhedrosis virus endochitinase like sequence; Chi: chitinase,^(b)anti-microbial peptide, ^(c)pre-hevein like protein, ^(d)hevein,^(e)chitin-binding protein, ^(f)pathogenesis related protein,^(g)wound-induced protein, ^(h)wheat germ agglutinin, ^(i)agglutinin(lectin). ¹References: 1) Udea et al. (1994) J. Ferment. Bioeng. 78,205-211 2) Watanabe et al. (1990) J. Biol. Chem. 265, 15659-16565 3)Watanabe et al. (1992) J. Bacteriol. 174, 408-414 4) Gleave et al.(1994) EMBL Data Library 5) Sidhu et al. (1994) J. Biol. Chem. 269,20167-20171 6) Jones et al. (1986) EMBO J. 5, 467-473 7) Sitrit et al.(1994) EMBL Data Library 8) Genbank entry only 9) Tsujibo et al. (1993)J. Bacteriol. 175, 176-181 10) Yanai et al. (1992) J. Bacteriol. 174,7398-7406 11) Pauley (1994) EMBL Data Library 12) Kuranda et al. (1991)J. Biol. Chem. 266, 19758-19767 13) van Damme et al. (1992) EMBL DataLibrary 14) Broekaert et al. (1992) Biochemistry 31, 4308-4314 15) deBolle et al. (1993) Plant Mol. Physiol. 22, 1187-1190 16) Samac et al.(1990) Plant Physiol. 93, 907-914 17) Potter et al. (1993) Mol. PlantMicrobe Interact. 6, 680-685 18) Buchanan-Wollaston (1995) EMBL DataLibrary 19) Hamel et al. (1993) Plant Physiol. 101, 1403-1403 20)Broekaert et al. (1990) Proc. Natl. Acad. Sci. USA 87, 7633-7637 21) Leeet al. (1991) J. Biol. Chem. 266, 15944-15948 22) Leah et al. (1994)Plant Physiol. 6, 579-589 23) Danhash et al. (1993) Plant Mol. Biol. 221017-1029 24) Ponstein et al. (1994) Plant Physiol. 104, 109-118 25)Meins et al. (1991) Patent EP0418695-A1 26) van Buuren et al. (1992)Mol. Gen. Genet. 232, 460-469 27) Shinshi et al. (1990) Plant Mol. Biol.14, 357-368 28) Cornellisen et al. (1991) Patent EP0440304-A2 29) Fukudaet al. (1991) Plant Mol. Biol. 16, 1-10 30) Yun et al. (1994) EMBL DataLibrary 31) Kim et al. (1994) Biosci. Biotechnol. Biochem. 58, 1164-116632) Nishizawa et al. (1993) Mol. Gen. Genet. 241, 1-10 33) Nishizawa etal. (1991) Plant Sci 76, 211-218 34) Huang et al. (1991) Plant Mol.Biol. 16, 479-480 35) Zhu et al. (1991) Mol. Gen. Genet. 226, 289-29636) Muthukrishhnan et al. (1993) EMBL Data Library 37) Xu (1995) EMBLData Library 38) Vad et al. (1993) Plant Sci 92, 69-79 39) Chang et al.(1994) EMBL Data Library 40) Davis et al. (1991) Plant Mol. Biol. 17,631-639 41) Clarke et al. (1994) Plant Mol. Biol. 25, 799-815 42)Broglie et al. (1989) Plant Cell 1, 599-607 43) Broglie et al. (1986)Proc. Natl. acad. Sci. USA 83, 6820-6824 44) Lucas et al. (1985) FEBSLett. 193, 208-210 45) Hedrick et al. (1988) Plant Physiol. 86, 182-18646) Roberts et al. (1994) EMBL Data LibraryI 47) Vamagami et al. (1994)Biosci. Biotechnol. Biochem. 58, 322-329 48) Beerhues et al. (1994)Plant Mol. Biol. 24, 353-367 49) Stanford et al. (1989) Mol. Gen. Genet.215, 200-208 50) Liao et al. (1993) EMBL Data Library 51) Smith et al.(1989) Plant Mol. Biol. 13, 601-603 52) Wright et al. (1989) J. Mol.Evol. 28, 327-336 53) Wright et al. (1984) Biochemistry 23, 280-287 54)Raikhel et al. (1987) Proc. Natl. acad. Sci. USA 84, 6745-6749 55)Hajela et al. (1993) EMBL Data Library 56) Lerner et al. (1992) J. Biol.Chem. 267, 11085-11091 57) Vo et al. (1995) EMBL Data Library

Table 8 herein below provides an overview of proteins containingStreptocooai glucan-binding repeats (Cpl superfamily) which may be usedas polysaccharide domains of the present invention.

TABLE 8 Overview of proteins containing Streptococcal glucan-bindingrepeats (Cpl superfamily) Source Protein Accession No. Ref.² S. downei(sobrinus) GTF-I D13858 1 (0MZ176) S. downei (sobrinus) GTF-IP11001/M17391 2 (MFe28) S. downei (sobrinus) GTF-S P29336/M30943/A414833 (MFe28) S. downei (sobrinus) (6715) GTF-I P27470/D90216/A38175 4 S.downei (sobrinus) DEI L34406 5 S. mutants (Ingbritt) GBP M30945/A37184 6S. mutants (GS-5) GTF-B A33128 7 S. mutants (GS-5) GTF-BP08987/M17361/B33135 8 S. mutants GTF- P05427/C33135 8 B^(3′-ORF) S.mutants (GS-5) GTF-C P13470/M17361/M22054 9 S. mutants (GS-5) GTF-C notavailable 10 S. mutants (GS-5) GTF-D M29296/A45866 11 S. salivariusGTF-J A44811/S22726/S28809 12 Z11873/M64111 S. salivarius GTF-KS22737/S22727/Z11872 13 S. salivarius (ATCC25975) GTF-L L35495 14 S.salivarius (ATCC25975) GTF-M L35928 14 S. pneumoniae R6 LytAP06653/A25634/M13812 15 S. pneumoniae PspA A41971/M74122 16 Phage HB-3HBL P32762/M34652 17 Phage Cp-1 CPL-1 P15057/J03586/A31086 18 Phage Cp-9CPL-9 P19386/M34780/JQ0438 19 Phage EJ-1 EJL A42936 20 C. difficile (VPI10463) ToxA P16154/A37052/M30307 21 X51797/S08638 C. difficile (BARTSW1) ToxA A60991/X17194 22 C. difficile (VPI 10463) ToxBP18177/X53138/X60984 23, 24 S10317 C. difficile (1470) ToxBS44271/Z23277 25, 26 C. novyi a-toxin S44272/Z23280 27 C. novyi a-toxinZ48636 28 C. acetobutylicum CspA S49255/Z37723 29 (NCIB8052) C.acetobutylicum CspB Z50008 30 (NCIB8052) C. acetobutylicum CspC Z5003330 (NCIB8052) C. acetobutylicum CspD Z50009 30 (NCIB8052)²References: 1) Sato et al. (1993) DNA sequence 4, 19-27 2) Ferreti etal. (1987) J. Bacteriol. 169, 4271-4278 3) Gilmore et al. (1990) J.Infect. Immun. 58, 2452-2458 4) Abo et al. (1991) J. Bacteriol. 173,989-996 5) Sun et al. (1994) J. Bacteriol. 176, 7213-7222 6) Banas etal. (1990) J. Infect. Immun. 58, 667-673 7) Shiroza et al. (1990)Protein Sequence Database 8) Shiroza et al. (1987) J. Bacteriol. 169,4263-4270 9) Ueda et al. (1988) Gene 69, 101-109 10) Russel (1990) Arch.Oral. Biol. 35, 53-58 11) Honda et al. (1990) J. Gen. Microbiol. 136,2099-2105 12) Giffard et al. (1991) J. Gen. Microbiol. 137, 2577-259313) Jacques (1992) EMBL Data Library 14) Simpson et al. (1995) J.Infect. Immun. 63, 609-621 15) Gargia et al. (1986) Gene 43, 265-272 16)Yother et al. (1992) J. Bacteriol. 174, 601-609 17) Romero et al. (1990)J. Bacteriol. 172, 5064-5070 18) Garcia et al. (1988) Proc. Natl. Acad.Sci, USA 85, 914-918 19) Garcia et al. (1990) Gene 86, 81-88 20) Diaz etal. (1992) J. Bacteriol. 174, 5516-5525 21) Dove et al. (1990) J.Infect. Immun. 58, 480-488 22) Wren et al. (1990) FEMS Microbiol. Lett.70, 1-6 23) Barroso et a. (1990) Nucleic Acids Res. 18, 4004-4004 24)von Eichel-Streiber et al. (1992) Mol. Gen. Genet. 233, 260-268 25)Sartinger et al. (1993) EMBL Data Library 26) von Eichel-Streiber et al.(1995) Mol. Microbiol. In Press 27) Hofmann et al. (1993) EMBL DataLibrary 28) Hofmann et al. (1995) Mol. Gen. Genet. In Press 29) Sanchezet al. (1994) EMBL Data Library 30) Sanchez et al. (1995) EMBL DataLibrary

Table 9 below lists proteins containing putative β-1,3 glucan-bindingdomains which may be contemplated as the polysaccharide binding domainsof the present invention.

TABLE 9 Overview of proteins containing putative Source (strain) Proteinaccession No. Ref³ Type I B. circulans (WL-12) GLCA1P23903/M34503/JQ0420 1 B. circulans (IAM 1165) BglH JN0772/D17519/S670332 Type II Actinomadura sp. (FC7) XynII U08894 3 Arthrobacter sp. (YCWD3)GLCI D23668 9 O. xanthineolytica GLC P22222/M60826/A39094 4 R.faecitabidus (YLM-50) RP I Q05308/A45053/D10753 5a,b R. communis RicinA12892 6 S. lividans (1326) XlnA P26514/M64551/ 7 JS07986 T. tridentatusFactorGa D16622 8 B.: Bacillus, O.: Oerskovia, R. faecitabidus:Rarobacter faecitabidus, R. communis: Ricinus communis, S.:Streptomyces, T.: Tachypleus (Horseshoe Crab) ³References: 1) Yahata etal. (1990) Gene 86, 113-117 2) Yamamoto et al. (1993) Biosci.Biotechnol. Biochem. 57, 1518-1525 3) Harpin et al. (1994) EMBL DataLibrary 4) Shen et al. (1991) J. Biol. Chem. 266, 1058-1063 5a) Shimoiet al. (1992) J. Biol. Chem. 267, 25189-25195 5b) Shimoi et al. (1992)J. Biochem 110, 608-613 6) Horn et al. (1989) Patent A12892 7) Sharecket al. (1991) Gene 107, 75-82 8) Seki et al. (1994) J. Biol. Chem. 269,1370-1374 9) Watanabe et al. (1993) EMBL Data Library

The term “polypeptide” as used herein encompasses native polypeptides(either degradation products, synthetically synthesized polypeptides orrecombinant polypeptides) and peptidomimetics (typically, syntheticallysynthesized polypeptides), as well as peptoids and semipeptoids whichare polypeptide analogs, which may have, for example, modificationsrendering the polypeptides more stable while in a body or more capableof penetrating into cells. Such modifications include, but are notlimited to N′ terminus modification, C′ terminus modification,polypeptide bond modification, including, but not limited to, CH2-NH,CH2-S, CH2-S═O, O═C—NH, CH2-O, CH2-CH2, S═C—NH, CH═CH or CF═CH, backbonemodifications, and residue modifications. Methods for preparingpeptidomimetic compounds are well known in the art and are specified,for example, in Quantitative Drug Design, C. A. Ramsden Gd., Chapter17.2, F. Choplin Pergamon Press (1992), which is incorporated byreference as fully set forth herein. Further details in this respect areprovided hereinunder.

Polypeptide bonds (—CO—NH—) within the polypeptide may be substituted,for example, by N-methylated bonds (—N(CH3)-CO—), ester bonds(—C(R)H—C—O—O—C(R)—N—), ketomethylen bonds (—CO—CH2-), α-aza bonds(—NH—N(R)—CO—), wherein R is any alkyl, e.g., methyl, carba bonds(—CH2-NH—), hydroxyethylene bonds (—CH(OH)—CH2-), thioamide bonds(—CS—NH—), olefinic double bonds (—CH═CH—), retro amide bonds (—NH—CO—),polypeptide derivatives (—N(R)—CH2-CO—), wherein R is the “normal” sidechain, naturally presented on the carbon atom.

These modifications can occur at any of the bonds along the polypeptidechain and even at several (2-3) at the same time.

Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted forsynthetic non-natural acids such as Phenylglycine,1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (TIC), naphthylelanine(Nol), ring-methylated derivatives of Phe, halogenated derivatives ofPhe or o-methyl-Tyr.

As mentioned, the amino acid sequences of polypeptides of fibrousproteins may either be the amino acid sequences of the polypeptides innaturally-occurring fibrous proteins or those that comprise eitherconservative or non-conservative substitutions.

The term “conservative substitution” as used herein, refers to thereplacement of an amino acid present in the native sequence in thepeptide with a naturally or non-naturally occurring amino or apeptidomimetics having similar steric properties. Where the side-chainof the native amino acid to be replaced is either polar or hydrophobic,the conservative substitution should be with a naturally occurring aminoacid, a non-naturally occurring amino acid or with a peptidomimeticmoiety which is also polar or hydrophobic (in addition to having thesame steric properties as the side-chain of the replaced amino acid).

As naturally occurring amino acids are typically grouped according totheir properties, conservative substitutions by naturally occurringamino acids can be easily determined bearing in mind the fact that inaccordance with the invention replacement of charged amino acids bysterically similar non-charged amino acids are considered conservativesubstitutions.

For producing conservative substitutions by non-naturally occurringamino acids it is also possible to use amino acid analogs (syntheticamino acids) well known in the art. A peptidomimetic of the naturallyoccurring amino acids is well documented in the literature known to theskilled practitioner.

When effecting conservative substitutions the substituting amino acidshould have the same or a similar functional group in the side chain asthe original amino acid.

The phrase “non-conservative substitutions” as used herein refers toreplacement of the amino acid as present in the parent sequence byanother naturally or non-naturally occurring amino acid, havingdifferent electrochemical and/or steric properties. Thus, the side chainof the substituting amino acid can be significantly larger (or smaller)than the side chain of the native amino acid being substituted and/orcan have functional groups with significantly different electronicproperties than the amino acid being substituted. Examples ofnon-conservative substitutions of this type include the substitution ofphenylalanine or cyclohexylmethyl glycine for alanine, isoleucine forglycine, or —NH—CH[(—CH₂)₅—COOH]—CO—for aspartic acid. Thosenon-conservative substitutions which fall within the scope of thepresent invention are those which still constitute a polypeptide beingable to form a fibrous protein.

As used herein in the specification and in the claims section below, theterm “amino acid” or “amino acids” is understood to include the 20naturally occurring amino acids; those amino acids often modifiedpost-translationally in vivo, including, for example, hydroxyproline,phosphoserine and phosphothreonine; and other unusual amino acidsincluding, but not limited to, 2-aminoadipic acid, hydroxylysine,isodesmosine, nor-valine, nor-leucine and ornithine. Furthermore, theterm “amino acid” includes both D- and L-amino acids.

Tables 10 and 11 below list naturally occurring amino acids (Table 10)and non-conventional or modified amino acids (Table 11) which can beused with the present invention.

TABLE 10 Three-Letter Amino Acid Abbreviation One-letter Symbol alanineAla A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys CGlutamine Gln Q Glutamic Acid Glu E glycine Gly G Histidine His Hisoleucine Iie I leucine Leu L Lysine Lys K Methionine Met Mphenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr Ttryptophan Trp W tyrosine Tyr Y Valine Val V Any amino acid as above XaaX

TABLE 11 Non-conventional amino acid Code Non-conventional amino acidCode α-aminobutyric acid Abu L-N-methylalanine Nmalaα-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmargaminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylateL-N-methylaspartic acid Nmasp aminoisobutyric acid AibL-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgincarboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine ChexaL-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucineNmile D-alanine Dal L-N-methylleucine Nmleu D-arginine DargL-N-methyllysine Nmlys D-aspartic acid Dasp L-N-methylmethionine NmmetD-cysteine Dcys L-N-methylnorleucine Nmnle D-glutamine DglnL-N-methylnorvaline Nmnva D-glutamic acid Dglu L-N-methylornithine NmornD-histidine Dhis L-N-methylphenylalanine Nmphe D-isoleucine DileL-N-methylproline Nmpro D-leucine Dleu L-N-methylserine Nmser D-lysineDlys L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophanNmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine DpheL-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine NmetgD-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine DthrL-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine Dtyrα-methyl-aminoisobutyrate Maib D-valine Dval α-methyl-γ-aminobutyrateMgabu D-α-methylalanine Dmala α ethylcyclohexylalanine MchexaD-α-methylarginine Dmarg α-methylcyclopentylalanine McpenD-α-methylasparagine Dmasn α-methyl-α-napthylalanine ManapD-α-methylaspartate Dmasp α-methylpenicillamine Mpen D-α-methylcysteineDmcys N-(4-aminobutyl)glycine Nglu D-α-methylglutamine DmglnN-(2-aminoethyl)glycine Naeg D-α-methylhistidine DmhisN-(3-aminopropyl)glycine Norn D-α-methylisoleucine DmileN-amino-α-methylbutyrate Nmaabu D-α-methylleucine Dmleu α-napthylalanineAnap D-α-methyllysine Dmlys N-benzylglycine Nphe D-α-methylmethionineDmmet N-(2-carbamylethyl)glycine Ngln D-α-methylornithine DmornN-(carbamylmethyl)glycine Nasn D-α-methylphenylalanine DmpheN-(2-carboxyethyl)glycine Nglu D-α-methylproline DmproN-(carboxymethyl)glycine Nasp D-α-methylserine Dmser N-cyclobutylglycineNcbut D-α-methylthreonine Dmthr N-cycloheptylglycine NchepD-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex D-α-methyltyrosineDmty N-cyclodecylglycine Ncdec D-α-methylvaline DmvalN-cyclododeclglycine Ncdod D-α-methylalnine Dnmala N-cyclooctylglycineNcoct D-α-methylarginine Dnmarg N-cyclopropylglycine NcproD-α-methylasparagine Dnmasn N-cycloundecylglycine NcundD-α-methylasparatate Dnmasp N-(2,2-diphenylethyl)glycine NbhmD-α-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine NbheD-N-methylleucine Dnmleu N-(3-indolylyethyl) glycine NhtrpD-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate NmgabuN-methylcyclohexylalanine Nmchexa D-N-methylmethionine DnmmetD-N-methylornithine Dnmorn N-methylcyclopentylalanine NmcpenN-methylglycine Nala D-N-methylphenylalanine DnmpheN-methylaminoisobutyrate Nmaib D-N-methylproline DnmproN-(1-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nleu D-N-methylthreonine DnmthrD-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine NvaD-N-methyltyrosine Dnmtyr N-methyla-napthylalanine NmanapD-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acidGabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine TbugN-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine PenL-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine MargL-α-methylasparagine Masn L-α-methylaspartate MaspL-α-methyl-t-butylglycine Mtbug L-α-methylcysteine McysL-methylethylglycine Metg L-α thylglutamine Mgln L-α-methylglutamateMglu L-α-methylhistidine Mhis L-α-methylhomo phenylalanine MhpheL-α-methylisoleucine Mile N-(2-methylthioethyl)glycine NmetD-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine NargD-N-methylglutamate Dnmglu N-(1-hydroxyethyl)glycine NthrD-N-methylhistidine Dnmhis N-(hydroxyethyl)glycine NserD-N-methylisoleucine Dnmile N-(imidazolylethyl)glycine NhisD-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine NhtrpD-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate NmgabuN-methylcyclohexylalanine Nmchexa D-N-methylmethionine DnmmetD-N-methylornithine Dnmorn N-methylcyclopentylalanine NmcpenN-methylglycine Nala D-N-methylphenylalanine DnmpheN-methylaminoisobutyrate Nmaib D-N-methylproline DnmproN-(1-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nleu D-N-methylthreonine DnmthrD-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine NvalD-N-methyltyrosine Dnmtyr N-methyla-napthylalanine NmanapD-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acidGabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine TbugN-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine PenL-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine MargL-α-methylasparagine Masn L-α-methylaspartate MaspL-α-methyl-t-butylglycine Mtbug L-α-methylcysteine McysL-methylethylglycine Metg L-α-methylglutamine Mgln L-α-methylglutamateMglu L-α ethylhistidine Mhis L-α-methylhomophenylalanine Mhphe L-αthylisoleucine Mile N-(2-methylthioethyl)glycine Nmet L-α-methylleucineMleu L-α-methyllysine Mlys L-α-methylmethionine MmetL-α-methylnorleucine Mnle L-α-methylnorvaline Mnva L-α-methylornithineMorn L-α-methylphenylalanine Mphe L-α-methylproline MproL-α-methylserine mser L-α-methylthreonine Mthr L-α ethylvaline MtrpL-α-methyltyrosine Mtyr L-α-methylleucine MvalL-N-methylhomophenylalanine Nmhphe

nbhm N-(N-(2,2-diphenylethyl) N-(N-(3,3-diphenylpropyl)carbamylmethyl-glycine Nnbhm carbamylmethyl(1)glycine Nnbhe1-carboxy-1-(2,2-diphenylnylamino)cyclopropane Nmbc

indicates data missing or illegible when filed

Amino acid sequences of exemplary polypeptides of the present inventionare set forth in SEQ ID NOs: 11-13, 55, 57, 58 and SEQ ID NOs. 32-36.

Recombinant techniques are preferably used to generate the polypeptidesof the present invention since these techniques are better suited forgeneration of relatively long polypeptides (e.g., longer than 20 aminoacids) and large amounts thereof. Such recombinant techniques aredescribed by Bitter et al., (1987) Methods in Enzymol. 153:516-544,Studier et al. (1990) Methods in Enzymol. 185:60-89, Brisson et al.(1984) Nature 310:511-514, Takamatsu et al. (1987) EMBO J. 6:307-311,Coruzzi et al. (1984) EMBO J. 3:1671-1680, Brogli et al., (1984) Science224:838-843, Gurley et al. (1986) Mol. Cell. Biol. 6:559-565 andWeissbach & Weissbach, 1988, Methods for Plant Molecular Biology,Academic Press, NY, Section VIII, pp 421-463.

To produce a polypeptide of the present invention using recombinanttechnology, a polynucleotide encoding a polypeptide of the presentinvention is ligated into a nucleic acid expression vector, whichcomprises the polynucleotide sequence under the transcriptional controlof a cis-regulatory sequence (e.g., promoter sequence) suitable fordirecting constitutive, tissue specific or inducible transcription ofthe polypeptides of the present invention in the host cells.

An example of an isolated polynucleotide which can be used to expressresilin is as set forth in SEQ ID NO: 15. Examples of isolatedpolynucleotide sequences which can be used to express spider silk are asset forth in SEQ ID NOs: 23 and 27. An example of an isolatedpolynucleotide which can be used to express a cellulose binding domainis set forth in SEQ ID NO: 25. Exemplary polynucleotide sequences whichcan be used to express the polypeptides of the present invention are setforth in SEQ ID NO: 17-22, 24, 28 and 29.

The phrase “an isolated polynucleotide” refers to a single or doublestranded nucleic acid sequence which is isolated and provided in theform of an RNA sequence, a complementary polynucleotide sequence (cDNA),a genomic polynucleotide sequence and/or a composite polynucleotidesequences (e.g., a combination of the above).

As used herein, the phrase “complementary polynucleotide sequence”refers to a sequence which results from reverse transcription ofmessenger RNA using a reverse transcriptase or any other RNA-dependentDNA polymerase. Such a sequence can be subsequently amplified in vivo orin vitro using a DNA-dependent DNA polymerase.

As used herein, the phrase “genomic polynucleotide sequence” refers to asequence derived (isolated) from a chromosome and thus represents acontiguous portion of a chromosome.

As used herein, the phrase “composite polynucleotide sequence” refers toa sequence, which is at least partially complementary and at leastpartially genomic. A composite sequence can include some exonalsequences required to encode the polypeptide of the present invention,as well as some intronic sequences interposing therebetween. Theintronic sequences can be of any source, including of other genes, andtypically will include conserved splicing signal sequences. Suchintronic sequences may further include cis-acting expression regulatoryelements.

The polynucleotides of the present invention may further comprise asignal sequence encoding a signal peptide for the secretion of thefibrous polypeptide. An exemplary signal sequence that may be used inthe constructs of the present invention (for plant transfection) is avacuolar signal sequence.

Following expression and secretion, the signal peptides are typicallyremoved from the precursor proteins resulting in the mature proteins.

Polynucleotides of the present invention may be prepared using PCRtechniques as described in Example 1 and Example 7 herein below, or anyother method or procedure known in the art for ligation of two differentDNA sequences. See, for example, “Current Protocols in MolecularBiology”, eds. Ausubel et al., John Wiley & Sons, 1992.

As mentioned hereinabove, polynucleotide sequences of the presentinvention are inserted into expression vectors (i.e., a nucleic acidconstruct) to enable expression of the recombinant polypeptide. Theexpression vector of the present invention includes additional sequenceswhich render this vector suitable for replication and integration inprokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors).Typical cloning vectors contain transcription and translation initiationsequences (e.g., promoters, enhances) and transcription and translationterminators (e.g., polyadenylation signals).

Eukaryotic promoters typically contain two types of recognitionsequences, the TATA box and upstream promoter elements. The TATA box,located 25-30 base pairs upstream of the transcription initiation site,is thought to be involved in directing RNA polymerase to begin RNAsynthesis. The other upstream promoter elements determine the rate atwhich transcription is initiated.

Enhancer elements can stimulate transcription up to 1,000-fold fromlinked homologous or heterologous promoters. Enhancers are active whenplaced downstream or upstream from the transcription initiation site.Many enhancer elements derived from viruses have a broad host range andare active in a variety of tissues. For example, the SV40 early geneenhancer is suitable for many cell types. Other enhancer/promotercombinations that are suitable for the present invention include thosederived from polyoma virus, human or murine cytomegalovirus (CMV), thelong term repeat from various retroviruses such as murine leukemiavirus, murine or Rous sarcoma virus and HIV. See, Enhancers andEukaryotic Expression, Cold Spring Harbor Press, Cold Spring Harbor,N.Y. 1983, which is incorporated herein by reference.

In the construction of the expression vector, the promoter is preferablypositioned approximately the same distance from the heterologoustranscription start site as it is from the transcription start site inits natural setting. As is known in the art, however, some variation inthis distance can be accommodated without loss of promoter function.

In addition to the elements already described, the expression vector ofthe present invention may typically contain other specialized elementsintended to increase the level of expression of cloned nucleic acids orto facilitate the identification of cells that carry the recombinantDNA. For example, a number of animal viruses contain DNA sequences thatpromote the extra chromosomal replication of the viral genome inpermissive cell types. Plasmids bearing these viral replicons arereplicated episomally as long as the appropriate factors are provided bygenes either carried on the plasmid or with the genome of the host cell.

The vector may or may not include a eukaryotic replicon. If a eukaryoticreplicon is present, then the vector is amplifiable in eukaryotic cellsusing the appropriate selectable marker. If the vector does not comprisea eukaryotic replicon, no episomal amplification is possible. Instead,the recombinant DNA integrates into the genome of the engineered cell,where the promoter directs expression of the desired nucleic acid.

The expression vector of the present invention can further includeadditional polynucleotide sequences that allow, for example, thetranslation of several proteins from a single mRNA such as an internalribosome entry site (IRES) and sequences for genomic integration of thepromoter-chimeric polypeptide.

Examples for mammalian expression vectors include, but are not limitedto, pcDNA3, pcDNA3.1(+/−), pGL3, pZeoSV2(+/−), pSecTag2, pDisplay,pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1,pNMT41, pNMT81, which are available from Invitrogen, pCI which isavailable from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which areavailable from Strategene, pTRES which is available from Clontech, andtheir derivatives.

Expression vectors containing regulatory elements from eukaryoticviruses such as retroviruses can be also used. SV40 vectors includepSVT7 and pMT2. Vectors derived from bovine papilloma virus includepBV-1MTHA, and vectors derived from Epstein Bar virus include pHEBO, andp2O5. Other exemplary vectors include pMSG, pAV009/A+, pMTO10/A+,pMAMneo-5, baculovirus pDSVE, and any other vector allowing expressionof proteins under the direction of the SV-40 early promoter, SV-40 laterpromoter, metallothionein promoter, murine mammary tumor virus promoter,Rous sarcoma virus promoter, polyhedrin promoter, or other promotersshown effective for expression in eukaryotic cells.

Viruses are very specialized infectious agents that have evolved, inmany cases, to elude host defense mechanisms. Typically, viruses infectand propagate in specific cell types. The targeting specificity of viralvectors utilizes its natural specificity to specifically targetpredetermined cell types and thereby introduce a recombinant gene intothe infected cell. Thus, the type of vector used by the presentinvention will depend on the cell type transformed.

Recombinant viral vectors may be useful for expression of thepolypeptides of the present invention since they offer advantages suchas lateral infection. Lateral infection is inherent in the life cycleof, for example, retrovirus and is the process by which a singleinfected cell produces many progeny virions that bud off and infectneighboring cells. The result is that a large area becomes rapidlyinfected, most of which was not initially infected by the original viralparticles. This is in contrast to vertical-type infection in which theinfectious agent spreads only through daughter progeny. Viral vectorscan also be produced that are unable to spread laterally. Thischaracteristic can be useful if the desired purpose is to introduce aspecified gene into only a localized number of targeted cells.

A variety of prokaryotic or eukaryotic cells can be used ashost-expression systems to express the polypeptides of the presentinvention. These include, but are not limited to, microorganisms, suchas bacteria (for example, E. coli including but not limited to E. colistrains BL21 (DE3) plysS, BL21; (DE3)RP and BL21* and B. subtilis)transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmidDNA expression vector containing the polypeptide coding sequence; yeasttransformed with recombinant yeast expression vectors containing thepolypeptide coding sequence; plant cell systems infected withrecombinant virus expression vectors (e.g., cauliflower mosaic virus,CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmidexpression vectors, such as Ti plasmid, containing the polypeptidecoding sequence.

Various methods can be used to introduce the expression vector of thepresent invention into the cells of the host expression system. Suchmethods are generally described in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Springs Harbor Laboratory, New York (1989,1992), in Ausubel et al., Current Protocols in Molecular Biology, JohnWiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic GeneTherapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al., GeneTargeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey ofMolecular Cloning Vectors and Their Uses, Butterworths, Boston Mass.(1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986] andinclude, for example, stable or transient transfection, lipofection,electroporation and infection with recombinant viral vectors. Inaddition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 forpositive-negative selection methods.

Introduction of nucleic acids by viral infection offers severaladvantages over other methods such as lipofection and electroporation,since higher transfection efficiency can be obtained due to theinfectious nature of viruses.

According to one embodiment, the polypeptides of the present inventionare expressed in plants.

The term “plant” as used herein encompasses whole plants, ancestors andprogeny of the plants and plant parts, including seeds, shoots, stems,roots (including tubers), and plant cells, tissues and organs. The plantmay be in any form including suspension cultures, embryos, meristematicregions, callus tissue, leaves, gametophytes, sporophytes, pollen, andmicrospores. Plants that are particularly useful in the methods of theinvention include all plants which belong to the superfamilyViridiplantee, in particular monocotyledonous and dicotyledonous plantsincluding a fodder or forage legume, ornamental plant, food crop, tree,or shrub selected from the list comprising Acacia spp., Acer spp.,Actinidia spp., Aesculus spp., Agathis australis, Albizia amara,Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, Asteliafragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp., Brassicaspp., Bruguiera gymnorrhiza, Burkea africana, Butea frondosa, Cadabafarinosa, Calliandra spp, Camellia sinensis, Canna indica, Capsicumspp., Cassia spp., Centroema pubescens, Chacoomeles spp., Cinnamomumcassia, Coffea arabica, Colophospermum mopane, Coronillia varia,Cotoneaster serotina, Crataegus spp., Cucumis spp., Cupressus spp.,Cyathea dealbata, Cydonia oblonga, Cryptomeria japonica, Cymbopogonspp., Cynthea dealbata, Cydonia oblonga, Dalbergia monetaria, Davalliadivaricata, Desmodium spp., Dicksonia squarosa, Dibeteropogonamplectens, Dioclea spp, Dolichos spp., Dorycnium rectum, Echinochloapyramidalis, Ehraffia spp., Eleusine coracana, Eragrestis spp.,Erythrina spp., Eucalypfus spp., Euclea schimperi, Eulalia vi/losa,Pagopyrum spp., Feijoa sellowlana, Fragaria spp., Flemingia spp,Freycinetia banksli, Geranium thunbergii, GinAgo biloba, Glycinejavanica, Gliricidia spp, Gossypium hirsutum, Grevillea spp., Guibourtiacoleosperma, Hedysarum spp., Hemaffhia altissima, Heteropogon contoffus,Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hypeffheliadissolute, Indigo incamata, Iris spp., Leptarrhena pyrolifolia,Lespediza spp., Lettuca spp., Leucaena leucocephala, Loudetia simplex,Lotonus bainesli, Lotus spp., Macrotyloma axillare, Malus spp., Manihotesculenta, Medicago saliva, Metasequoia glyptostroboides, Musasapientum, Nicotianum spp., Onobrychis spp., Ornithopus spp., Oryzaspp., Peltophorum africanum, Pennisetum spp., Persea gratissima, Petuniaspp., Phaseolus spp., Phoenix canariensis, Phormium cookianum, Photiniaspp., Picea glauca, Pinus spp., Pisum sativam, Podocarpus totara,Pogonarthria fleckii, Pogonaffhria squarrosa, Populus spp., Prosopiscineraria, Pseudotsuga menziesii, Pterolobium stellatum, Pyrus communis,Quercus spp., Rhaphiolepsis umbellata, Rhopalostylis sapida, Rhusnatalensis, Ribes grossularia, Ribes spp., Robinia pseudoacacia, Rosaspp., Rubus spp., Salix spp., Schyzachyrium sanguineum, Sciadopitysvefficillata, Sequoia sempervirens, Sequoiadendron giganteum, Sorghumbicolor, Spinacia spp., Sporobolus fimbriatus, Stiburus alopecuroides,Stylosanthos humilis, Tadehagi spp, Taxodium distichum, Themedatriandra, Trifolium spp., Triticum spp., Tsuga heterophylla, Vacciniumspp., Vicia spp., Vitis vinifera, Watsonia pyramidata, Zantedeschiaaethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli, Brusselssprouts, cabbage, canola, carrot, cauliflower, celery, collard greens,flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean,straw, sugar beet, sugar cane, sunflower, tomato, squash tea, trees.Alternatively, algae and other non-Viridiplantae can be used for themethods of the present invention.

It will be appreciated that in order to express the polypeptides of thepresent invention in plants, the constructs encoding same typicallycomprise a plant-expressible promoter.

As used herein, the phrase “plant-expressible” refers to a promotersequence, including any additional regulatory elements added thereto orcontained therein, is at least capable of inducing, conferring,activating or enhancing expression in a plant cell, tissue or organ,preferably a monocotyledonous or dicotyledonous plant cell, tissue, ororgan. One exemplary promoter that may be useful in the constructs ofthe present invention is the RbcS1 promoter (SEQ ID NO: 30), either inaddition to or in absence of SEQ ID NO: 31, as exemplified in theExamples section herein under. Of note, other sequences may also be usedfor plant expression such as set forth in SEQ ID NOs: 48 and 50.

Nucleic acid sequences of the polypeptides of the present invention maybe optimized for plant expression. Examples of such sequencemodifications include, but are not limited to, an altered G/C content tomore closely approach that typically found in the plant species ofinterest, and the removal of codons atypically found in the plantspecies commonly referred to as codon optimization.

The phrase “codon optimization” refers to the selection of appropriateDNA nucleotides for use within a structural gene or fragment thereofthat approaches codon usage within the plant of interest. Therefore, anoptimized gene or nucleic acid sequence refers to a gene in which thenucleotide sequence of a native or naturally occurring gene has beenmodified in order to utilize statistically-preferred orstatistically-favored codons within the plant. The nucleotide sequencetypically is examined at the DNA level and the coding region optimizedfor expression in the plant species determined using any suitableprocedure, for example as described in Sardana et al. (1996, Plant CellReports 15:677-681). In this method, the standard deviation of codonusage, a measure of codon usage bias, may be calculated by first findingthe squared proportional deviation of usage of each codon of the nativegene relative to that of highly expressed plant genes, followed by acalculation of the average squared deviation. The formula used is: 1SDCU=n=1N [(Xn−Yn)/Yn]2/N, where Xn refers to the frequency of usage ofcodon n in highly expressed plant genes, where Yn to the frequency ofusage of codon n in the gene of interest and N refers to the totalnumber of codons in the gene of interest. A table of codon usage fromhighly expressed genes of dicotyledonous plants is compiled using thedata of Murray et al. (1989, Nuc Acids Res. 17:477-498).

One method of optimizing the nucleic acid sequence in accordance withthe preferred codon usage for a particular plant cell type is based onthe direct use, without performing any extra statistical calculations,of codon optimization tables such as those provided on-line at the CodonUsage Database through the NIAS (National Institute of AgrobiologicalSciences) DNA bank in Japan (www.kazusa.or.jp/codon/). The Codon UsageDatabase contains codon usage tables for a number of different species,with each codon usage table having been statistically determined basedon the data present in Genbank.

By using the above tables to determine the most preferred or mostfavored codons for each amino acid in a particular species (for example,rice), a naturally-occurring nucleotide sequence encoding a protein ofinterest can be codon optimized for that particular plant species. Thisis effected by replacing codons that may have a low statisticalincidence in the particular species genome with corresponding codons, inregard to an amino acid, that are statistically more favored. However,one or more less-favored codons may be selected to delete existingrestriction sites, to create new ones at potentially useful junctions(5′ and 3′ ends to add signal peptide or termination cassettes, internalsites that might be used to cut and splice segments together to producea correct full-length sequence), or to eliminate nucleotide sequencesthat may negatively effect mRNA stability or expression.

The naturally-occurring encoding nucleotide sequence may already, inadvance of any modification, contain a number of codons that correspondto a statistically-favored codon in a particular plant species.Therefore, codon optimization of the native nucleotide sequence maycomprise determining which codons, within the native nucleotidesequence, are not statistically-favored with regards to a particularplant, and modifying these codons in accordance with a codon usage tableof the particular plant to produce a codon optimized derivative. Amodified nucleotide sequence may be fully or partially optimized forplant codon usage provided that the protein encoded by the modifiednucleotide sequence is produced at a level higher than the proteinencoded by the corresponding naturally occurring or native gene.Construction of synthetic genes by altering the codon usage is describedin for example PCT Patent Application 93/07278.

Thus, the present invention encompasses nucleic acid sequences describedhereinabove; fragments thereof, sequences hybridizable therewith,sequences homologous thereto, sequences orthologous thereto, sequencesencoding similar polypeptides with different codon usage, alteredsequences characterized by mutations, such as deletion, insertion orsubstitution of one or more nucleotides, either naturally occurring orman induced, either randomly or in a targeted fashion.

Exemplary polynucleotide sequences that may be used to express thepolypeptides of the present invention in plants are set forth in SEQ IDNOs: 20-22.

Plant cells may be transformed stably or transiently with the nucleicacid constructs of the present invention. In stable transformation, thenucleic acid molecule of the present invention is integrated into theplant genome and as such it represents a stable and inherited trait. Intransient transformation, the nucleic acid molecule is expressed by thecell transformed but it is not integrated into the genome and as such itrepresents a transient trait.

There are various methods of introducing foreign genes into bothmonocotyledonous and dicotyledonous plants (Potrykus, I., Annu. Rev.Plant. Physiol., Plant. Mol. Biol. (1991) 42:205-225; Shimamoto et al.,Nature (1989) 338:274-276).

The principle methods of causing stable integration of exogenous DNAinto plant genomic DNA include two main approaches:

(i) Agrobacterium-mediated gene transfer: Klee et al. (1987) Annu. Rev.Plant Physiol. 38:467-486; Klee and Rogers in Cell Culture and SomaticCell Genetics of Plants, Vol. 6, Molecular Biology of Plant NuclearGenes, eds. Schell, J., and Vasil, L. K., Academic Publishers, SanDiego, Calif. (1989) p. 2-25; Gatenby, in Plant Biotechnology, eds.Kung, S, and Arntzen, C. J., Butterworth Publishers, Boston, Mass.(1989) p. 93-112.

(ii) direct DNA uptake: Paszkowski et al., in Cell Culture and SomaticCell Genetics of Plants, Vol. 6, Molecular Biology of Plant NuclearGenes eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego,Calif. (1989) p. 52-68; including methods for direct uptake of DNA intoprotoplasts, Toriyama, K. et al. (1988) Bio/Technology 6:1072-1074. DNAuptake induced by brief electric shock of plant cells: Zhang et al.Plant Cell Rep. (1988) 7:379-384. Fromm et al. Nature (1986)319:791-793. DNA injection into plant cells or tissues by particlebombardment, Klein et al. Bio/Technology (1988) 6:559-563; McCabe et al.Bio/Technology (1988) 6:923-926; Sanford, Physiol. Plant. (1990)79:206-209; by the use of micropipette systems: Neuhaus et al., Theor.Appl. Genet. (1987) 75:30-36; Neuhaus and Spangenberg, Physiol. Plant.(1990) 79:213-217; glass fibers or silicon carbide whiskertransformation of cell cultures, embryos or callus tissue, U.S. Pat. No.5,464,765 or by the direct incubation of DNA with germinating pollen,DeWet et al. in Experimental Manipulation of Ovule Tissue, eds. Chapman,G. P. and Mantell, S. H. and Daniels, W. Longman, London, (1985) p.197-209; and Ohta, Proc. Natl. Acad. Sci. USA (1986) 83:715-719.

Although stable transformation is presently preferred, transienttransformation of leaf cells, meristematic cells or the whole plant isalso envisaged by the present invention.

Transient transformation can be effected by any of the direct DNAtransfer methods described above or by viral infection using modifiedplant viruses.

Viruses that have been shown to be useful for the transformation ofplant hosts include CaMV, TMV and BV. Transformation of plants usingplant viruses is described in U.S. Pat. No. 4,855,237 (BGV), EP-A 67,553(TMV), Japanese Published Application No. 63-14693 (TMV), EPA 194,809(BV), EPA 278,667 (BV); and Gluzman, Y. et al., Communications inMolecular Biology: Viral Vectors, Cold Spring Harbor Laboratory, NewYork, pp. 172-189 (1988). Pseudovirus particles for use in expressingforeign DNA in many hosts, including plants, is described in WO87/06261.

Independent of the host cell system, it will be appreciated that otherthan containing the necessary elements for the transcription andtranslation of the inserted coding sequence (encoding the polypeptide),the expression construct of the present invention can also includesequences engineered to optimize stability, production, purification,yield or activity of the expressed polypeptide.

Transformed cells are cultured under effective conditions, which allowfor the expression of high amounts of recombinant polypeptide. Effectiveculture conditions include, but are not limited to, effective media,bioreactor, temperature, pH and oxygen conditions that permit proteinproduction. An effective medium refers to any medium in which a cell iscultured to produce the recombinant polypeptide of the presentinvention. Such a medium typically includes an aqueous solution havingassimilable carbon, nitrogen and phosphate sources, and appropriatesalts, minerals, metals and other nutrients, such as vitamins. Cells ofthe present invention can be cultured in conventional fermentationbioreactors, shake flasks, test tubes, microtiter dishes and petriplates. Culturing can be carried out at a temperature, pH and oxygencontent appropriate for a recombinant cell. Such culturing conditionsare within the expertise of one of ordinary skill in the art.

Depending on the vector and host system used for production, resultantpolypeptides of the present invention may either remain within therecombinant cell, secreted into the fermentation medium, secreted into aspace between two cellular membranes, such as the periplasmic space inE. coli; or retained on the outer surface of a cell or viral membrane.

Following a predetermined time in culture, recovery of the recombinantpolypeptide is effected.

The phrase “recovering the recombinant polypeptide” used herein refersto collecting the whole fermentation medium containing the polypeptideand need not imply additional steps of separation or purification.

Thus, polypeptides of the present invention can be purified using avariety of standard protein purification techniques, such as, but notlimited to, affinity chromatography, ion exchange chromatography,filtration, electrophoresis, hydrophobic interaction chromatography, gelfiltration chromatography, reverse phase chromatography, concanavalin Achromatography, chromatofocusing and differential solubilization.

To facilitate recovery, the expressed coding sequence can be engineeredto encode the polypeptide of the present invention and fused cleavablemoiety e.g. histidine. Such a fusion protein can be designed so that thepolypeptide can be readily isolated by affinity chromatography; e.g., byimmobilization on a column specific for the cleavable moiety. Examples3-5 and 8 describe purification of resilin and spidersilk polypeptidesof the present invention.

Where a cleavage site is engineered between the polypeptide and thecleavable moiety, the polypeptide can be released from thechromatographic column by treatment with an appropriate enzyme or agentthat specifically cleaves the fusion protein at this site [e.g., seeBooth et al., Immunol. Lett. 19:65-70 (1988); and Gardella et al., J.Biol. Chem. 265:15854-15859 (1990)].

The polypeptide of the present invention is preferably retrieved in a“substantially pure” form.

As used herein, the phrase “substantially pure” refers to a purity thatallows for the effective use of the protein in the applicationsdescribed herein.

In addition to being synthesizable in host cells, the polypeptide of thepresent invention can also be synthesized using in vitro expressionsystems. These methods are well known in the art and the components ofthe system are commercially available.

Following expression and optional purification of the polypeptides ofthe present invention, the polypeptides may be polymerized to form aninsoluble material from a solution, preferably one with a relativelyhigh concentration of polypeptide. According to one embodiment, thecritical concentration of a resilin polypeptide of the present inventionis about 50 mg/ml. According to one embodiment, the polypeptide isconcentrated by ultracentrifugation.

Generally, crosslinking of proteins can be performed using standardcrosslinking agents such as gluteraldahyde, di-isocyanate and Genipin.Exemplary polymerization conditions for particular fibrous polypeptidemonomers are presented herein below.

Crosslinking Conditions for Resilin:

According to a preferred embodiment, the crosslinking is such thatdityrosine bonds are formed. These methods are well known to the personskilled in the art and are discussed by Malencik and Anderson(Biochemistry 1996, 35, 4375-4386), the contents of which areincorporated herein by reference.

In an embodiment, enzyme-mediated cross-linking in the presence ofRu(bpy)3Cl2.6; H2O may be employed. Exemplary peroxidases that may beused to crosslink resilin include, but are not limited to horseradishperoxidase, Arthromyces peroxidase, Duox peroxidase from Caenorhabditiselegans, Sea urchin ovoperoxidases and Chorion.

Following irradiation, a Ru(III) ion is formed, which serves as anelectron abstraction agent to produce a carbon radical within thepolypeptide, preferentially at a tyrosine residue, and thus allowsdityrosine link formation. This method of induction allows quantitativeconversion of soluble resilin or pro-resilin fragments to a very highmolecular weight aggregate. Moreover this method allows for convenientshaping of the bioelastomer by introducing recombinant resilin into aglass tube of the desired shape and irradiating the recombinant resilincontained therein.

In another embodiment, UV irradiation is effected in order to crosslinkthe resilin polypeptides of the present invention [Lehrer S S, Fasman GD. (1967) Biochemistry. 6(3):757-67; Malencik D A, Anderson S R. (2003)Amino Acids. 25(3-4):233-47], although care must be taken not to damagethe protein through exposure to this radiation. UVB radiationcross-linking may also be undertaken in the presence or absence ofriboflavin. In the absence of riboflavin, a substantial amount ofcross-linking takes place within one hour of exposure. The crosslinkingtime is substantially reduced if riboflavin is present. Still further,cross-linking may be effected with ultra-violet light in the presence ofcoumarin or by white light in the presence of fluorescein. An analysisof the dityrosine may be performed using conventional methods such ashigh performance liquid chromatography measurements in order toascertain the extent of dityrosine cross-link formation.

Metal ions and H2O2 may also be used to induce dytirosine formationthrough Fenton's reaction [Ali F E, J Inorg Biochem. 98(1):173-84].

Crosslinking Conditions for Elastin:

Following heating above the transition temperature (Tm), elastin may becrosslinked using the following oxidizing agents: lysil oxidasebis(sulfosuccimidyl) suberate, pyrroloquinoline quinine (PQQ),catechol/peroxidase reagent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the presence of N-hydroxybenzotriazole,N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC),1-hydroxybenzotriazole hydrate (HOBt); 1,6-diisocyanatohexane (HOBt);glutaraldehyde; N-hydroxysuccinimide (NHS), genipin.

Elastin may also be crosslinked by γ-irradiation, or, followingfunctionalization with methacrylate, it may also be photo-crosslinked.

Crosslinking Conditions for Silk:

Silk polypeptides, such as spider silk and silkworm silk may bepolymerized into β sheets using organic solvents, such as methanol.Alternatively, the silk polypeptides may be solubilized in waterfollowed by dehydration in order to form β sheets.

Crosslinking Conditions for Collagen:

Collagen may be crosslinked by glutaraldehyde and other chemicalcrosslinking agents, by glycation using different sugars, by Fentonreaction using metal ions such as cupper, by lysine oxidase or by UVradiation.

To determine the effect of cross-links and the optimal number ofcross-links per monomer unit, the resilience of a cross-linked polymercan be measured using methods known in the art. The level ofcross-linking can vary provided that the resulting polymer displays therequisite resilient properties. For example, when the cross-linking isby gamma-irradiation, the degree of cross-linking is a function of thetime and energy of the irradiation. The time required to achieve adesired level of cross-linking may readily be computed by exposingnon-cross-linked polymer to the source of radiation for different timeintervals and determining the degree of resilience (elastic modulus) ofthe resulting cross-linked material for each time interval. By thisexperimentation, it will be possible to determine the irradiation timerequired to produce a level of resiliency appropriate for a particularapplication.

The extent of cross-linking may be monitored during the reaction orpre-determined by using a measured amount of reactants. For example, inthe case of resilin polypeptides, since-the dityrosine cross-link isfluorescent, the fluorescence spectrum of the reactant mixture may bemonitored during the course of a reaction to determine the extent ofcross-linking at any particular time. Once the desired level ofcross-linking is achieved (indicated by reaching a predeterminedfluorescence intensity) a peroxidase-catalysed reaction may be quenchedby for example the addition of glutathione.

The polypeptides of the present invention may be used as are or they maybe blended with polysaccharides in order to generate novel compositematerials.

Thus, according to another aspect of the present invention, there isprovided an isolated composite comprising a fibrous polypeptide and apolysaccharide.

As used herein the term “composite” refers to a a substantially solidmaterial that is composed of two or more discrete materials, one beingthe fibrous polypeptide, the other the polysaccharide, each of whichretains its identity, e.g., physical characteristics, while contributingdesirable properties to the composite.

The term “isolated” as used herein refers to the composite beingsubstantially free from other substances (e.g., other cells, proteins,nucleic acids, etc.) in its in-vivo environment (e.g in the case ofresilin-chitin composites, removed from other insect wing components).According to another embodiment, the composites are also isolated from(i.e. removed from) solid supports (i.e. are non-immobilized).

Exemplary polysaccharides contemplated for the composites of the presentinvention include, but are not limited to chitin, cellulose, starch,dextran, glucan, chitosan, alginate and hyaluronic acid.

The cellulose may be in the form of powder such as Sigmacell, cellulosewhiskers, cellulose threads or 3D structures such as paper or scaffolds.Whisker preparation is typically performed by hydrolysis of cellulosewith 60% H2SO2 for 1 to 6 hours at 60° C. followed by sonication. Thesuspension is then diluted in double distilled H2O (DDW) followed byrepeated cycles (at least 5) of resuspension with DDW and centrifugationto remove the acid. Finally, the whiskers pellet is dialyzed against DDWwhile monitoring the pH up to 7. Whiskers quality may be monitored byTransfer Electron Microscopy (TEM).

According to one embodiment of this aspect of the present invention, themonomers of the fibrous polypeptides of the present invention comprisepolysaccharide binding domains (e.g. heterologous polysaccharide bindingdomains). Such polysaccharide binding domains allows directional bindingbetween the polysaccharide and the fibrous polypeptide at defined pointsof contact. Furthermore, the affinity of the fibrous polypeptide for thepolysaccharide may be adjusted according to the polysaccharide bindingdomain.

Other composites which are contemplated by the present invention includethose comprising two fibrous polypeptides wherein at least one of whichcomprises a heterologous polysaccharide binding domain.

Such composites may also comprise polysaccharides. Thus composites oftwo fibrous polypeptides and a polysaccharide are also contemplated bythe present invention.

It is expected that the composites of the present invention compriseenhanced characteristics (e.g. increased strength) compared to theconstituent fibrous polypeptide since the flat and ordered surface ofthe polysaccharide (e.g. cellulose whisker) may serve as a template forassembly of the fibrous polypeptide that usually requires shearing andelongation stress.

In order to generate the composites of the present invention,suspensions of monomers of the fibrous polypeptides and thepolysaccharides (e.g. cellulose whiskers)—for example at approximately2% solid content, are blended together.

Exemplary ratio of the component suspension include: 100/0, 90/10,80/20, 70/30, 60/40, 50/50, 40/60, 30/70, 20/80, 10/90, and 0/100.

The mixed solutions may be cast onto suitable molds (e.g. Teflon orpolystyrene) following which appropriate assembly and crosslinking isoptionally effected.

As mentioned above, the type of crosslinking depends on the fibrouspolypeptide of the composite. The crosslinking may be effected in thepresence of other fibrous polypeptides to generate the two fibrouspolypeptide/polysaccharide composites described herein above.

The present invention also contemplates coating the novel composites.According to one embodiment, the coating is comprised of fibrouspolypeptides. In this method, following the crosslinking of thecomposites, dipping in solutions of other fibrous polypeptides may becarried out. The fibrous proteins in the coating will typically beabsorbed into the composite. Following coating, a suitablepolymerization method may be used depending on the actual fibrouspolypeptide of the coating. For example, a cellulose-resilin compositemay be dipped in a solution containing silk monomers. Subsequently thecomposite may be transferred into 90% methanol solution which promotessilk β-sheet formation resulting in a cellulose-resilin-silk compositematerial.

The composites of the present invention may be combined with otherpolymers in blends and adducts to manipulate the degradation andmechanical properties of the material. Practically any biocompatiblepolymer may be combined with the composites. In a preferred embodiment,the added polymer is biodegradable. Exemplary biodegradable polymersinclude natural polymers and their synthetic analogs, includingpolysaccharides, proteoglycans, glycosaminoglycans, collagen-GAG,collagen, fibrin, and other extracellular matrix components, such aselastin, fibronectin, vitronectin, and laminin. Hydrolyticallydegradable polymers known in the art include, for example, certainpolyesters, polyanhydrides, polyorthoesters, polyphosphazenes, andpolyphosphoesters. Biodegradable polymers known in the art, include, forexample, certain polyhydroxyacids, polypropylfumerates,polycaprolactones, polyhydroxyalkanoates, poly (amide-enamines),polyamides, poly (amino acids), polyacetals, polyethers, biodegradablepolycyanoacrylates, biodegradable polyurethanes and polysaccharides. Forexample, specific biodegradable polymers that may be used in the presentinvention include but are not limited to, polylysine, poly (lactic acid)(PLA), poly (glycolic acid) (PGA), copolymers and mixtures of PLA andPGA, e.g., poly (lactide-co-glycolide) (PLG), poly (caprolactone) (PCL),poly (lactide-co-caprolactone) (PLC), and poly(glycolide-co-caprolactone) (PGC).

Those skilled in the art will recognize that this is an exemplary, notcomprehensive, list of biodegradable polymers. The properties of theseand other polymers and methods for preparing them are further describedin the art. See, for example, U.S. Pat. Nos. 6,123,727; 5,804,178;5,770,417; 5,736,372; 5,716,404 to Vacanti; U.S. Pat. Nos. 6,095,148;5,837,752 to Shastri; U.S. Pat. No. 5,902,599 to Anseth; U.S. Pat. Nos.5,696,175; 5,514,378; 5,512,600 to Mikos; U.S. Pat. No. 5,399,665 toBarrera; U.S. Pat. No. 5,019,379 to Domb; U.S. Pat. No. 5,010,167 toRon; U.S. Pat. Nos. 4,806,621; 4,638,045 to Kohn; and U.S. Pat. No.4,946,929 to d'Amore; see also Wang et al., J. Am. Chem. Soc. 123: 9480,2001; Lim et al., J. Am. Chem. Soc. 123: 2460, 2001; Langer, Acc. Chen7.Res. 33: 94, 2000; Langer, J. Control Release 62: 7, 1999; and Uhrich etal., Chem. Rev. 99: 3181, 1999.

The composites of the present invention may also be combined withnon-biodegradable polymers. For example, polypyrrole, polyanilines,polythiophene, and derivatives thereof are useful electricallyconductive polymers that can provide additional stimulation to seededcells or neighboring tissue. Exemplary non-biodegradable polymersinclude, but are not limited to, polystyrene, polyesters,non-biodegradable polyurethanes, polyureas, poly (ethylene vinylacetate), polypropylene, polymethacrylate, polyethylene, polycarbonates,and poly (ethylene oxide).

The importance of biopolymer based biomaterials is constantly increasingin the field of reconstructive medicine. In the recent years, the focusof this field has turned from the search for inert materials forimplantation to development of biopolymer based materials that interactwith the tissue and promote its correct regeneration. Furthermore,synthetic implants often fail the test of long term biocompatibilityrequiring their replacement during the lifetime of a patient which is amajor drawback. With respect to reconstructive medicine, polysaccharidesand protein polymers have been extensively investigated.

When used in vivo, and in particular inside the body of a subject, e.g.,a human patient, it is important that the composites of the presentinvention be biocompatible. A “biocompatible” material is notsubstantially mutagenic, antigenic, inflammatory, pyrogenic, orhemolytic. Furthermore, it must neither exhibit substantialcytotoxicity, acute systemic toxicity, or intracutaneous toxicity, norsignificantly decrease clotting time. In vivo and in vitro tests forthese undesirable biological activities are well known in the art;examples of such assays are given, for example, in U.S. Pat. No.5,527,610, the contents of which are incorporated by reference. Also,when used in vivo, the materials may be biogradable.

In the event that toxicity or immunogenicity, for example, occurs in arelevant composite, methods for modulating these undesirable effects areknown in the art. For example, “tanning” of the composite by treating itwith chemicals such as aldehydes (e.g., glutaraldehyde) or metaperiodatewill substantially decrease both toxicity and immunogenicity.Preferably, the composites used to make devices for in vivo use are alsosterilizable.

As mentioned, the composites of the present invention may be used in thefield of reconstructive medicine such as for the generation ofscaffolds.

As used herein, the term “scaffold” refers to a 3D matrix upon whichcells may be cultured (i.e., survive and preferably proliferate for apredetermined time period).

The scaffold may be fully comprised of the composites of the presentinvention, or may comprise a solid support on which is layered thecomposites of the present invention.

A “solid support,” as used refers to a three-dimensional matrix or aplanar surface (e.g. a cell culture plate) on which cells may becultured. The solid support can be derived from naturally occurringsubstances (i.e., protein based) or synthetic substances. Suitablesynthetic matrices are described in, e.g., U.S. Pat. Nos. 5,041,138,5,512,474, and 6,425,222. For example, biodegradable artificialpolymers, such as polyglycolic acid, polyorthoester, or polyanhydridecan be used for the solid support. Calcium carbonate, aragonite, andporous ceramics (e.g., dense hydroxyapatite ceramic) are also suitablefor use in the solid support. Polymers such as polypropylene,polyethylene glycol, and polystyrene can also be used in the solidsupport.

Therapeutic compounds or agents that modify cellular activity can alsobe incorporated (e.g. attached to, coated on, embedded or impregnated)into the scaffold composite material or a portion thereof. In addition,agents that act to increase cell attachment, cell spreading, cellproliferation, cell differentiation and/or cell migration in thescaffold may also be incorporated into the scaffold. Such agents can bebiological agents such as an amino acid, peptides, polypeptides,proteins, DNA, RNA, lipids and/or proteoglycans.

Suitable proteins which can be used along with the present inventioninclude, but are not limited to, extracellular matrix proteins [e.g.,fibrinogen, collagen, fibronectin, vimentin, microtubule-associatedprotein 1D, Neurite outgrowth factor (NOF), bacterial cellulose (BC),laminin and gelatin], cell adhesion proteins [e.g., integrin,proteoglycan, glycosaminoglycan, laminin, intercellular adhesionmolecule (ICAM) 1, N-CAM, cadherin, tenascin, gicerin, RGD peptide andnerve injury induced protein 2 (ninjurin2)], growth factors [epidermalgrowth factor, transforming growth factor-α, fibroblast growthfactor-acidic, bone morphogenic protein, fibroblast growth factor-basic,erythropoietin, thrombopoietin, hepatocyte growth factor, insulin-likegrowth factor-I, insulin-like growth factor-II, Interferon-β,platelet-derived growth factor, Vascular Endothelial Growth Factor andangiopeptin], cytokines [e.g., M-CSF, IL-1beta, IL-8,beta-thromboglobulin, EMAP-II, G-CSF and IL-10], proteases [pepsin, lowspecificity chymotrypsin, high specificity chymotrypsin, trypsin,carboxypeptidases, aminopeptidases, proline-endopeptidase,Staphylococcus aureus V8 protease, Proteinase K (PK), aspartic protease,serine proteases, metalloproteases, ADAMTS17, tryptase-gamma, andmatriptase-2] and protease substrates.

Additionally and/or alternatively, the scaffolds of the presentinvention may comprise an antiproliferative agent (e.g., rapamycin,paclitaxel, tranilast, Atorvastatin and trapidil), an immunosuppressantdrug (e.g., sirolimus, tacrolimus and Cyclosporine) and/or anon-thrombogenic or anti-adhesive substance (e.g., tissue plasminogenactivator, reteplase, TNK-tPA, glycoprotein IIb/IIIa inhibitors,clopidogrel, aspirin, heparin and low molecular weight heparins such asenoxiparin and dalteparin).

The scaffolds of the present invention may be administered to subjectsin need thereof for the regeneration of tissue such as connectivetissue, muscle tissue such as cardiac tissue and pancreatic tissue.Examples of connective tissues include, but are not limited to,cartilage (including, elastic, hyaline, and fibrocartilage), collagen,adipose tissue, reticular connective tissue, embryonic connectivetissues (including mesenchymal connective tissue and mucous connectivetissue), tendons, ligaments, and bone.

The composites of the present invention may thus be used for treating acartilage or bone disease or condition.

Exemplary cartilage conditions include, but are not limited toosteoarthritis, limited joint mobility, gout, rheumatoid arthritis,osteoarthritis, chondrolysis, scleroderma, degenerative disc disorderand systemic lupus erythematosus.

As used herein, the term “treating” refers to inhibiting or arrestingthe development of a disease, disorder or condition and/or causing thereduction, remission, or regression of a disease, disorder or conditionin an individual suffering from, or diagnosed with, the disease,disorder or condition. Those of skill in the art will be aware ofvarious methodologies and assays which can be used to assess thedevelopment of a disease, disorder or condition, and similarly, variousmethodologies and assays which can be used to assess the reduction,remission or regression of a disease, disorder or condition.

As used herein, the term “subject” refers to mammals, including, but notlimited to, humans, canines and horses.

It will be appreciated that the composites of the present inventioncomprises a myriad of medical uses other than for tissue regenerationand for treating cartilage and bone diseases including, but not limitedto treatment of urinary incontinence (e.g. urethral bulking), as ahealing aid for burn patients and as a dressing to prevent bleeding.

In addition, other medical applications may also benefit from theelasticity, biodegradability and/or biovavailabiliy of the composites ofthe present invention. For example, after abdominal surgery, theintestines and other abdominal organs tend to adhere to one another andto the abdominal wall. It is thought that this adhesion results frompost-surgical inflammation, however, anti-inflammatory drugs delivereddirectly to the abdominal region dissipate quickly. The composites ofthe present invention (e.g. those comprising resilin) may be used todeliver anti-inflammatory drugs to the abdominal region.

A soft and flexible composite may be implanted between the abdominalwall and internal organs, for example, by attaching it to the abdominalwall, without cutting internal organs, which would lead to infection.The anti-inflammatory drug can be released from the composite over aperiod of months. While previous researchers have attempted to usehydrogels, hyaluronic acid-based membranes, and other materials to solvethese problems, such materials tend to degrade quickly in the body; alonger resident period is necessary to prevent adhesion.

In another embodiment, the composites of the present invention may beused to coat a metallic stent. Because the composites may be madeflexible, they will expand with the stent without ripping, while thestiffness of the metal stent will prevent the composites fromelastically assuming its previous shape. The composites being highlybioavailable may release heparin or other anti-coagulants oranti-inflammatory agents to prevent the formation of clots or scartissue, which could close off the blood vessel or throw off a thrombusthat could cause circulatory problems, including stroke, elsewhere inthe body. Alternatively or in addition, angiogenic agents may be used topromote the remodeling of the blood vessel surrounding the stent.Indeed, any biomolecule, small molecule, or bioactive agent may becombined with the composites of the present invention. Such moleculesmay be covalently or non-covalently linked with the composites.

The composites of the present invention may also be used to prepare“long term” medical devices. Unlike typical permanent medical devices,the composites of the present invention will degrade over time. Forexample, the material may be fabricated into a biodegradable cardiacstent. Preferably, the composites are combined with a harder polymerthat plastically forms for the production of stents. Exemplary polymersinclude any of the polymers listed above, preferably biodegradablepolymers. The bio-rubber acts as a plasticizer that enables the stent toexpand into the desired shape after implantation. The stent increasesthe diameter of the blood vessel to allow easier circulation, but,because the stent is biodegradable, surrounding blood vessels increasein diameter without thrombosis or covering the stent with scar tissue,which would reclose the blood vessel. The time the stent should remainin place and retain its shape before degradation will vary from patientto patient and depend partially on the amount of blockage and the age ofthe patient (e.g., older patients require more time to heal). Oneskilled in the art will easily be able to adjust the molecular weightand cross-link density of the composites in the stent to adjust thedegradation rate. As for the coated stent, the degradable stent mayrelease biomolecules, small molecules, bioactive agents, or somecombination of these in situ.

The composites of the present invention may also be used to support invivo sensors and catheters. The composites may be constructed into achamber for an optical fiber-based sensor or a coating for a catheterthat is inserted into the area of interest. In a sensor, the chambercontains a specific chromophore-bonded receptor for the molecule ofinterest. When an analyte attaches to the receptor, the chromophore willeither emit or absorb light at an specific wavelength. The absorption oremission may be detected by an apparatus connected to the optical fiber.The sensor may be used for short term, continuous monitoring, forexample, for ten to fifteen days. Likewise, a catheter may be used toperiodically deliver drugs or other small molecules or bioactive agentsto a specific site or intravenously. Use of biodegradable composites ofthe present invention reduces the formation of scar tissue which wouldordinarily form around a shunt or other implant that is used for morethan two weeks. The degradation rate of the composite should beoptimized so that there is not significant degradation of the materialwhile it is in place in the patient.

The composites of the present invention may also be used for otherwounds that are hard to close or that fail to heal properly throughnormal physiologic mechanisms. For example, diabetics often get skininjuries (“diabetic ulcers”), especially in the lower extremities, whichtake a long time to heal or fail to heal properly due to poorcirculation. The use of the present composites to deliver antibiotics oranti-inflammatory agents to these wounds will aid healing and provide acover for the wound.

Other implantable medical devices which may be fabricated from thecomposites of the present invention include artificial blood vessels,catheters and other devices for the removal or delivery of fluids topatients, artificial hearts, artificial kidneys, orthopedic pins, platesand implants; catheters and other tubes (including urological andbiliary tubes, endotracheal tubes, peripherably insertable centralvenous catheters, dialysis catheters, long term tunneled central venouscatheters peripheral venous catheters, short term central venouscatheters, arterial catheters, pulmonary catheters, Swan-Ganz catheters,urinary catheters, peritoneal catheters), urinary devices (includinglong term urinary devices, tissue bonding urinary devices, artificialurinary sphincters, urinary dilators), shunts (including ventricular orarterio-venous shunts); prostheses (including breast implants, penileprostheses, vascular grafting prostheses, aneurysm repair devices, heartvalves, artificial joints, artificial larynxes, otological implants),anastomotic devices, vascular catheter ports, clamps, embolic devices,wound drain tubes, hydrocephalus shunts, pacemakers and implantabledefibrillators, and the like.

Of note, the cellulose produced by Gluconacetobacter xylinus is mostsuitable for medical applications. The Bacterial cellulose (BC) producedby these bacteria has high mechanical strength combined with negligibleforeign body and inflammatory responses that make it an attractivematerial for development of medical applications. BC has excellent waterretaining properties which make it suitable for production of chronicwound burn dressings and even artificial skin. Furthermore BC and BCcomposites can be shaped into almost any desired three-dimensionalstructure.

The composites of the present invention may be formulated aspharmaceutical and/or cosmetic compositions.

The term “cosmetic composition” as used herein refers to a compositionformulated for external application to human or animal skin, nails, orhair for the purpose of beautifying, coloring, conditioning, orprotecting the body surface. The present cosmetic composition can be inany form including for example: a gel, cream, lotion, makeup, coloredcosmetic formulations, shampoo, hair conditioner, cleanser, toner,aftershave, fragrance, nail enamel, and nail treatment product.

The phrase “colored cosmetic formulation” refers to cosmetics containingpigment including for example eye shadow, lipsticks and glosses, lip andeye pencils, mascara, and blush.

As mentioned, the composites of the present invention may also be usedas cosmetic agents for treatment of skin and hair.

Thus, the present invention contemplates the composites (e.g. comprisingcollagen) of the present invention as a substance which can be topicallyapplied, optionally in combination with other active substance such asfor example a vitamin (vitamin A, C, E or their mixtures) or othertopically active substances including but not limited to avarol, avaroneor plant extracts, such as Extr. Cepae or Extr. Echinaceae pallidae. Thecomposites of the present invention may be formulated as topical agentsin the form of creams, ointments, lotions or gels such as a hydrogelse.g. on the basis of polyacrylate or an oleogel e.g. made of water andEucerin.

Oleogels comprising both an aqueous and a fatty phase are basedparticularly on Eucerinum anhydricum, a basis of wool wax alcohols andparaffin, wherein the percentage of water and the basis can vary.Furthermore additional lipophilic components for influencing theconsistency can be added, e.g. glycerin, polyethylene glycols ofdifferent chain length, e.g. PEG400, plant oils such as almond oil,liquid paraffin, neutral oil and the like. The hydrogels of the presentinvention can be produced through the use of gel-forming agents andwater, wherein the first are selected especially from natural productssuch as cellulose derivatives, such as cellulose ester and ether, e.g.hydroxyethyl-hydroxypropyl derivatives, e.g. tylose, or also fromsynthetic products such as polyacrylic acid derivatives, such asCarbopol or Carbomer, e.g. P934, P940, P941. They can be produced orpolymerized based on known regulations, from alcoholic suspensions byadding bases for gel formation.

The cosmetic compositions may comprise other agents capable ofconditioning the body surface including, for example humectants;emollients; oils including for example mineral oil; and shine enhancersincluding for example dimethicone and cyclomethicone. The presentconditioning agents may be included in any of the presentpharmacological and/or cosmetic compositions.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Herein the term “active ingredient” refers to the collagen accountablefor the biological effect.

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, especially transnasal, intestinal or parenteraldelivery, including intramuscular, subcutaneous and intramedullaryinjections as well as intrathecal, direct intraventricular,intracardiac, e.g., into the right or left ventricular cavity, into thecommon coronary artery, intravenous, inrtaperitoneal, intranasal, orintraocular injections.

Alternately, one may administer the pharmaceutical composition in alocal rather than systemic manner, for example, via injection of thepharmaceutical composition directly into a tissue region of a patient.Thus, for treatment of urinary incontinence, the compositions of thepresent invention may be administered directly to the area surroundingthe urethra. For treatment of cartilage diseases, the compositions ofthe present invention may be administered by intra-articularadministration via a joint (e.g. directly into the knee, elbow, hip,sternoclavicular, temporomandibular, carpal, tarsal, wrist, ankle,intervertebral disk or a ligamentum flavum. For disc replacement, thepharmaceutical compositions of the present invention may als beadministered directly into the pulposus.

According to a particular embodiment of this aspect of the presentinvention, the composites of the present invention may be administereddirectly into the discs for total disc replacement, total disc nucleuspulposus replacement or disc nucleus polposus augmentation and repair ordirectly into the breast for breast augmentation. According to thisembodiment, the composites may be comprised in injectablenon-crosslinked formulations. Following injections of such formulations,photopolymerization may be initiated in situ. This may be effected usingclassical crosslinking techniques including gluteraldehyde, orcrosslinking via sugar molecules.

According to one embodiment, in-situ crosslinking of the injectableformulation may be affected by addition of an appropriate buffer (e.g.PBS pH 7.4) together with 200 M of CuCl2 and 10 mM of H2O2 so as togenerate dityrosine formation.

According to another embodiment, in situ crosslinking is effected usingthe same components described herein above, but the pH is maintained at5.2. This leads to modification of the tyrosines into DOPA. Followinginjection of the materials 0.1-0.5 mM of Sodioum periodate may be addedto form DOPA-DOPA bridges resulting in crossliniking of the fibrouspolypeptides.

According to another embodiment, in situ crosslinking is effected usingtyrosine crosslinking techniques involving H2O2 and radiation of theinjected material with UV.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can beformulated readily by combining the active compounds withpharmaceutically acceptable carriers well known in the art.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide.

The pharmaceutical composition described herein may be formulated forparenteral administration, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multidose containers with optionally, anadded preservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe active ingredients to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use.

The pharmaceutical composition of the present invention may also beformulated in rectal compositions such as suppositories or retentionenemas, using, e.g., conventional suppository bases such as cocoa butteror other glycerides.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount ofactive ingredients (composite) effective to prevent, alleviate orameliorate symptoms of a disorder (e.g. cartilage or bone disease).

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro and cell culture assays. For example, a dose can be formulatedin animal models to achieve a desired concentration or titer. Suchinformation can be used to more accurately determine useful doses inhumans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basisof Therapeutics”, Ch. 1 p. 1).

Dosage amount and interval may be adjusted individually to providetissue levels of the active ingredient are sufficient to induce orsuppress the biological effect (minimal effective concentration, MEC).The MEC will vary for each preparation, but can be estimated from invitro data. Dosages necessary to achieve the MEC will depend onindividual characteristics and route of administration. Detection assayscan be used to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA approved kit, which may containone or more unit dosage forms containing the active ingredient. The packmay, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser may also be accommodated by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions or human or veterinary administration. Suchnotice, for example, may be of labeling approved by the U.S. Food andDrug Administration for prescription drugs or of an approved productinsert. Compositions comprising a preparation of the inventionformulated in a compatible pharmaceutical carrier may also be prepared,placed in an appropriate container, and labeled for treatment of anindicated condition, as is further detailed above.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a polyeptide” or “at least one polypeptide” may include aplurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find support inthe following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a nonlimiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader. All the information contained therein is incorporated herein byreference.

Example 1 Construction of Resilin Chimeric Genes Preparation of ResilincDNA

According to Elvin et al [Nature. 437: 999-1002, 2005] resilin is mostlyexpressed at the pupa level in D. melanogaster. Therefore, RNA wasextracted from this stage for cDNA preparation. RNA was extracted fromD. melanogaster pupas using TRI® Reagent (Sigma, St. Louis, Mo.).Reverse transcription of the resilin cDNA was performed with M-MLV RT(H—) (Promega corporation, Madison Wis.) with oligo(dT)15 primeraccording to the manufacturers instructions.

Construction of Resilin fusion proteins: Four resilin genes weredesigned for expression in E. coli;

Resilin 17 elastic repeats including the native putative chitin bindingdomain (gi|45550440, nucleotides 698-1888) referred to as Res-ChBD gene.(Protein sequence: SEQ ID NO: 11, 55; polynucleotide sequence: SEQ IDNO:17)

Resilin 17 elastic repeats and the native linker for N-terminal fusionsand sole expression of a gene similar to Elvin et al, [Nature. 437:999-1002, 2005] (nucleotides 698-1666) referred to as Resilin (Proteinsequence: SEQ ID NO: 14, 56; polynucleotide sequence: SEQ ID NO: 15).

Clostridium cellulovorans CBD (CBDclos) fused to Resilin 17 elasticrepeats referred to as CBD-resilin (Protein sequence: SEQ ID NO: 12, 57;polynucleotide sequence: SEQ ID NO: 18).

Resilin (Gene No. 2) fused to CBD, referred to as Resilin-CBD (Proteinsequence: SEQ ID NO: 13, 58; polynucleotide sequence: SEQ ID NO: 19).

PCR primers were designed in order to construct the genes mentionedherein above as detailed in Table 12, herein below. A standard PCRmethod was designed suitable for all reactions: 94° C. for 4 minutes, 35cycles of 94° C. for 1 minute, 56° C. for 1 minute, 72° C. for 1 minuteand 72° C. for 4 minutes. All DNA products were separated on a 1%agarose gel. Appropriate bands were excised with a scalpel and the DNAwas purified with HiYield™ Gel/PCR DNA extraction kit (RBC Taipei,Taiwan).

TABLE 12 Primer Tm° No name Sequence Description C. 1 resCBD1.1CCATGGGACCGGAG Forward primer of 55 CCACCAGTTAACTC resilin + NcoI(SEQ ID NO: 1) site 2 resCHBDRev GGATCCTTAAGGAC Reverse primer of 57CGCTGGGACCACTG resilin + chitin (SEQ ID NO: 2) binding domain +BamHI site 3 resbmh1_ GGATCCCTCATCGT Reverse primer of 56 revTATCGTAGTCAGCG resilin 17 re- (SEQ ID NO: 3) peats + BamHIsite for N-term- inal fusion 4 CBD6P11 GTCTAGAAATAATT Forward primer of56 TTGTTTAACTTTAA CBD using pET- GAAGGAG CSCP37 as temp- (SEQ ID NO: 4)late + XbaI site 5 CBDRes2 AACTGGTGGCTCCG Reverse primer of 56GCATATCAAATGTT CBD + resilin GCAGAAGTAGGATT overhang (pink) AATTATTGfor PCR fusion (SEQ ID NO: 5) cloning 6 CBDRes3 TTCTGCAACATTTGForward primer of 56 ATCCGGAGCCACCA resilin + CBD GTTAACTCoverhang (blue) (SEQ ID NO: 6) for PCR fusion cloning 7 CBDRes4GGATCCTTACTCAT Reverse primer of 56 CGTTATCGTAGTCA resilin 17 re- GCGpeats + stop co- (SEQ ID NO: 7) don and BamHIsite

Construction of Res-ChBD:

Res-ChBD was the first gene that was constructed directly from the cDNAand served as PCR template for cloning of all the other resilin genes.The PCR was performed according to Table 13 herein below. Ex Taq™(Takara, Madison Wis.) is a proofreading enzyme suitable for TA cloning.

TABLE 13 Ingredient Volume (μl) TaKaRa Ex Taq ™ (5 units/μl) 0.25 10x ExTaq Buffer (Mg²⁺ plus) 5 dNTP Mixture (2.5 mM each) 4 Resilin cDNA 1Primer 1 resCBD1.1 (10 μmol) 1 Primer 2 resCHBDRev (10 μmol) 1Sterilized distilled water Up to 50

The 1200 base pair product (FIG. 1B) was purified and cloned into pGEM-TEasy vector (Promega Corporation, Madison Wis.). The presence ofresilin-ChBD was verified by sequencing. The sequencing was performedusing T7 and Sp6 primers that are complimentary to pGEM-T Easy vector.The sequencing results confirmed the cloning of the two resilin variantsaccording to Ardelll and Anderson [Insect Biochem Mol Biol. 31: 965-70,2002]. Variant A was chosen for further work.

Finally, the Res-ChBD was digested with NcoI, NotI restriction enzymesand cloned into pHis-parallel3 vector (FIG. 2) which contains anN-terminal His tag and a rTev cleavage site enabling purification of theprotein on Ni-NTA column and removal of the His tag if desired.

Construction of CBD-Resilin:

This gene was constructed by PCR-fusion method [Hobert O. (2002)Biotechniques. 32: 728-30]. A pET-CSCP vector [Levy et al., 2004,Biomaterials 25: 1841-1849] was used as template for CBD amplificationby PCR and the Res-ChBD product described herein above was used astemplate for resilin amplification. In the first round, two separatePCRs were performed. The CBD was amplified using primers No. 4 & 5. Theresilin was amplified with primers 6 & 7. The first amplification wasperformed with Deep VentR DNA Polymerase (NEB Inc. Ipswich, Mass.). Bythe end of the reactions, 1 l of each product (FIG. 3A) were mixed toserve as a template for the second step PCR. In this step, primers 4 and7 were used. The PCR was performed under the same conditions except theusage of Ex Taq™ (Takara, Madison Wis.) to allow TA cloning. The 1600base pair product was purified and cloned into pBluescript SK+(Ferments,Md.) (FIG. 3B). The presence of CBD-resilin was verified by sequencingwith T7 and T3 primers. The complete gene was digested with NcoI andNotI enzymes and cloned into pHis-parallel3 vector.

Construction of Resilin-CBD and Resilin (Genes 2 & 4):

The resilin gene was amplified using primers No. 1 and 3. The enzymeused for amplification was PfuTurbo® (Stratagene corporation, LA JollaCalif.). The PCR mixture used for generating DNA encoding Resilin-CBDand Resilin (genes 2 and 4) is described in Table 14, herein below.

TABLE 14 Ingredient Volume (μl) PfuTurbo ® (Stratagene corporation, 1 LAJolla CA) 10x cloned Pfu DNA polymerase reaction buffer 5 dNTP Mixture(2.5 mM each) 1 pGEM-T-ResCHBD (10 ng/μl) 1 Primer 1 resCBD1.1 (10 μmol)1 Primer 3 resbmh1_rev (10 μmol) 1 Sterilized distilled water Up to 30

Following the PCR reaction described herein above, 7 l of 10× Taqpolymerase reaction buffer, 1 l of Taq polymerase (Bio-lab, Israel), 1 lof dNTP mixture and sterilized distilled water to a volume of 100 l wasadded to the reaction tube. The tube was then incubated at 72° C. for 30minutes in order to add A nucleotides to the PCR product. The finalproduct was purified and cloned into pGEM-T Easy vector (Promegacorporation, Madison Wis.). The presence of the resilin gene wasverified by sequencing as described above.

For construction of Resilin-CBD gene, the resilin fragment was digestedwith NcoI, BamHI and cloned into pET3d (Novagen, EMD Chemicals, Inc. CA)upstream to the CBDclos gene followed by digestion of the Resilin-CBDwith NcoI, EcoRI and cloning into pHis-parallel3 vector digested withthe same enzymes.

Resilin expression vector (gene 2) was constructed by digestion ofpGEM-T Easy-Resilin with NcoI, NotI. In this way a stop codon was addedto the gene that allowed its direct expression. The gene wassubsequently cloned into pHis-parallel3 digested with the same enzymes.

Example 2 Expression of Resilin Chimeric Genes

All four vectors were transformed into BL21(DE3) (Novagen, EMDChemicals, Inc. CA). 5 ml of overnight cultures were grown in LB mediumwith 100 mg/L ampicillin at 37° C. rotary shaker. These starters wereused for inoculation of 250 to 350 ml of LB with 100 mg/L ampicillin ata ratio of 1/100 of starter to culture volume. At O.D. 600 of 0.8 to 0.9expression was induced with 1 mM IPTG. Four hours after induction,bacteria were harvested by centrifugation. 6H-Res-ChBD pellet wasdivided to 50 ml aliquots for initial analysis and the pellets werestored at −80° C.

Example 3 Purification of Resilin-ChBD and Characterization Thereof

Small Scale Batch Purification of 6H-Res-ChBD:

Bacterial pellet of 50 ml was re-suspended in 2 ml of 100 mM Tris pH7.5, 0.1% Triton® X-100, Complete™ (Roche, Basel Switzerland). Bacteriawere lyzed by sonication with pulsed bursts for 2 minutes on ice. Thesoluble and bacterial precipitates were separated by centrifugation at15000 RPM for 10 minutes at 4° C. SDS-PAGE analysis revealed that theRes-ChBD product is mostly found in the soluble fraction (FIG. 4 lanes1, 2). 500 μl of lysate were added into 1.5 ml eppendorf tube containing75 μl pre-equilibrated HIS-Select® Nickel Affinity Gel, (Sigma, St.Louis, Mo.). Purification was performed according to the product manual.Final elution was repeated twice with 100 μl elution buffer containing0.4 M imidazole.

Binding Assay of Purified 6H-Res-ChBD to Cellulose and Chitin:

25 mg of chitin (Sigma) and 50 mg of cellulose (Sigmacell) were added totwo separate 1.5 ml eppendorf tubes. The materials were washed with PBSfollowed by addition of 50 l of affinity purified protein solution. 450l of PBS were added to each tube to a total reaction volume of 500 l. Athird tube containing chitin only was supplemented with 500 l of PBS asnegative control since practical grade chitin (Sigma Cat No. C7170) thatcontained proteins was used. The tubes were incubated under gentlespinning for 30 minutes at RT followed by centrifugation. Thesupernatant was removed (unbound fraction) and the pellets were washed 3times with 500 l of PBS. The final pellets were boiled with 50 l of 2×sample application buffer (SAB). Samples of unbound and wash fractionsfrom each tube were also boiled with SAB. Samples were loaded on 12.5%SDS-PAGE gel.

Binding Assay of Crude Extracts of 6H-Res-ChBD to Cellulose and Chitin:

Bacterial lysates were produced from 50 ml pellets as described above.Cellulose and chitin binding assays were performed with 3 increasinglysate volumes as described in Table 15 below, in 2 ml eppendorf tubes.

TABLE 15 Lysate Tube No. volume (μl) 10x PBS (μl) DDH₂O (μl)Carbohydrate 1 50 50 400 Cellulose 50 mg 2 125 50 325 Cellulose 50 mg 3250 50 200 Cellulose 50 mg 4 50 50 400 Chitin 25 mg 5 125 50 325 Chitin25 mg 6 250 50 200 Chitin 25 mg

6H-Res-ChBD Thermostability Assay:

15 μl of affinity purified protein were added to 3 0.5 ml eppendorftubes. The tubes were incubated at 85° C. for 15, 30, 60 minutes. By theend of the incubation the tubes were transferred to ice and centrifugedat 14000 rpm for 10 minutes. Subsequently, the samples were boiled with2×SAB and loaded on 12.5% SDS-PAGE gel.

Small-Scale FPLC Purification of 6H-Res-ChBD:

Bacterial lysates were produced from 50 ml pellets as described above.The lysate was filtered with a syringe filter of 0.45 m for the purposeof FPLC (GE, Uppsala Sweden) purification on HisTrap™ HP (GE, UppsalaSweden) Ni-NTA 1 ml column pre-equilibrated according to the usermanual.

The purification program was run as follows:Binding buffer; 20 mM NaHPO4, 0.5 M NaCl, 10 mM imidazoleElution buffer; 20 mM NaHPO4, 0.5 M NaCl, 0.5 M imidazole

-   1. 5 column volumes (CV) of binding buffer at 1 ml/min.-   2. 5 ml injection of the lysate at 1 ml/min-   3. 5 CV wash with the binding buffer.-   4. linear gradient up to 500 mM imidazole for 10 min at 0.7 ml/min    with the elution buffer-   5. Equilibration with 5 CV of binding buffer at 1 ml/min.    Eluted proteins were detected at O.D.280. 400 l fractions were    collected and 10 l of samples boiled with SAB were loaded on a 12.5%    SDS-PAGE gel.

Production of Soluble High Molecular Weight 6H-Res-ChBD:

FPLC fractions 9 to 18 were collected to a total volume of 2 ml andimidazole was removed by three dialyses against 200 ml of polymerizationbuffer; 15 mM NaH₂PO₄, 150 mM NaCl pH 7.5. 500 μl of dialyzed proteinwas incubated at 85° C. for 10 min followed by O.N. incubation at 4° C.Polymerization was performed by adding 20 μl of 40 mM ammoniumpersulfate and 20 μl of 0.5 mM Ru(bpy)3Cl₂.6H₂O (Sigma) dissolved in thepolymerization buffer to an Eppendorf tube containing 40 μl of thepurified protein. The samples were subjected to sun light for 5 minutesfollowed by boiling with 2×SAB. Samples were loaded on 12.5% SDS-PAGEgel.

Medium-Scale FPLC Purification of 6H-Res-ChBD:

Bacterial pellets from 200 ml culture were resuspended in 15 ml of lysisbuffer as described above. Bacteria were lyzed by sonication with pulsedbursts for 5 minutes in an ice bath. The soluble and bacterialprecipitates were separated by centrifugation at 15000 RPM for 10minutes at 4° C. The purification was performed with FPLC using the samemethod as described above. Eluted proteins were detected at O.D.280. 400μl fractions were collected and 10 μl of samples boiled with SAB wereloaded on a 12.5% SDS-PAGE gel.

Production of Solid 6H-Res-ChBD:

Following medium-scale FPLC purification described herein above, thefractions were collected into two different dialysis bags; concentratedfractions No. 4 to 7 (6 ml) and diluted fractions No. 3, 8-12. Thedialysis was performed as described above. The concentration of theconcentrated peak was 10 mg/ml by O.D. 280 nm measurement. The samplewas loaded on a Vivaspin 6 10,000 MWCO (Sartorius Stedim Biotech,Aubagne, France) ultrafiltration tube and centrifuged at 5000 g for 40minutes. The final product gave around 500 μl at protein concentrationof 160 mg/ml. 40 μl of concentrated protein were pipette into aneppendorf tube that was added 4 μl of 250 mM ammonium persulfate and 1μl of 0.5 mM Ru(bpy)₃Cl₂.6H₂O. Immediately following the exposure of thetube to sunlight a solid polymer formed in the tube. The reaction wasstopped after 5 minutes by washing the polymer with water when no morepolymerization could be observed.

Results

Small-Scale Batch Purification of 6H-Res-ChBD:

Purification was effected as illustrated in FIG. 4 lanes 3-7.

Binding Assay of Purified 6H-Res-ChBD to Cellulose and Chitin:

Coomassie blue staining of the proteins revealed that 6H-Res-ChBD bindsboth to chitin and cellulose with a higher affinity to chitin (FIG. 5).The presence of protein in the unbound fraction is explained due thesaturation chitin/cellulose with Res-ChBD protein.

Binding Assay of Crude Extracts of 6H-Res-ChBD to Cellulose and Chitin:

Coomassie blue staining of the gels revealed that no cellulose bindingwas detected in crude lysates comprising the 6H-Res-ChBD (FIG. 6A, 6Blanes 2-5), contrasting the binding results following affinitychromatography AC purification. Nevertheless, the affinity of theprotein to chitin remained high as displayed by the crude lysatesresults. At 50 and 125 μl of crude lysate loaded on 25 mg of chitin,nearly 100% of the protein precipitated and very little protein remainedin the unbound fraction (FIG. 6B lanes 7-10, FIG. 6C lanes 2-5). When250 μl of lysate were applied, the amount of bound protein continued toincrease but a larger band was detected in the unbound fraction probablydue to saturation of the binding sites (FIG. 6C lanes 6-9).

6H-Res-ChBD Thermostability Assay:

Heat treatment displayed that 6H-Res-ChBD is stable at 85° C. for 1 hour(FIG. 7). As indicated in the Materials and Methods, the proteins wereimmediately transferred to ice following the heat treatment. This couldexplain the band shift observed in the gel due to initiation ofcoacervation process.

Small-Scale FPLC Purification of 6H-Res-ChBD:

The results of the purification process are illustrated in FIGS. 8A-B.

Production of Soluble High Molecular Weight 6H-Res-ChBD:

The results of the solubilization process are illustrated in FIG. 9.

Medium-Scale FPLC Purification of 6H-Res-ChBD:

The results of the purification process are illustrated in FIGS. 10A-B.

Example 4 Expression and Purification of 6H-Resilin 17 Elastic Repeatswithout any Polysaccharide Binding Domain (PBD) (SEQ ID NO: 56)

Following expression of the resilin of SEQ ID NO: 56 in E. coli, thesoluble protein was purified on a Ni-NTA column as illustrated in FIG.11. In addition, the protein was found to be thermostable and waspolymerized into solid resilin in the same manner as resilin-ChBD.

Example 5 Purification of CBD-Resilin (SEQ ID NO: 57) andCharacterization Thereof

Following expression of CBD-resilin in bacteria, it was found to beexpressed in inclusion bodies (FIG. 12).

Cells were lyzed by sonication in 0.1% Triton® X-100, Complete™ (Roche,Basel Switzerland).The insoluble fraction was precipitated by centrifugation.The supernatant was removed and the inclusion bodies were washed asfollows:

-   1. Resuspension with PBS buffer, 1% Triton® X-100, 1 mM EDTA, for 30    minutes with gentle shaking followed by centrifugation.-   2. Resuspension with PBS buffer, 1% Triton® X-100, for 30 minutes    with gentle shaking followed by centrifugation.-   3. Resuspension with PBS buffer for 30 minutes with gentle shaking    followed by centrifugation.    From that stage, purification of the inclusion bodies was performed    by one of two methods.-   1. Ni-NTA purification under denaturizing conditions. IBs were    solubilzed in 20 mM phosphate buffer pH 7.5, 20 mM imidazole, 0.5 M    NaCl, 6 M GuHCl. The proteins were loaded on pre-equilibrated Ni-NTA    column and the proteins were eluted with a linear gradient of 20 mM    phosphate buffer pH 7.5, 0.5 M imidazole, 0.5 M NaCl, 6 M GuHCl. The    fractions containing the peak that was detected at O.D. 280 nm were    collected and were refolded by dialysis against 50 mM Tris pH 7.5    buffer. The proteins were analyzed by SDS-PAGE. Refolding of the    protein was assayed by cellulose binding assay (FIG. 13).-   2. Washed IB were solubilzed in 20 mM phosphate buffer pH 7.5, 20 mM    imidazole, 0.5 M NaCl, 6 M GuHCl. The proteins were then injected to    the ÄKTAprime™ plus (GE Healthcare, Uppsala Sweden) loaded with    Ni-NTA column and purified using an automated refolding protocol    that is programmed in the machine. The fractions containing the    refolded proteins were collected (FIG. 14) followed by cellulose    binding assay. The automated refolded CBD-resilin protein was found    mostly in the bound fraction similar to the proteins refolded via    standard protocols, involving dialysis of samples purified in the    presence of 6M GuHCl or 8M urea, indicating that this method can be    applied since it is highly efficient and time saving.

Example 6 Cloning and Expression of Resilin-CBD (SEQ ID NO: 58)

A DNA fragment coding for resilin 17 elastic repeats+putative resilinlinker was cloned upstream to a vector containing the CBD to generate apolynucleotide of SEQ ID NO: 19. The correct insertion was verified bysequence followed by cloning of the gene into pHis parallel3 for proteinexpression. Expression was performed in BL21 bacteria similarly to allthe other proteins. Following protein expression the bacteria werecentrifuged and lyzed as described for CBD-resilin. The soluble andinsoluble fractions were separated by centrifugation. SDS-PAGE analysisrevealed that about 50% of the recombinant protein was found in thesoluble fraction. A cellulose binding assay was performed directly onresilin-CBD crude lysates resulting in high affinity binding ofresilin-CBD to cellulose (see FIG. 17).

Example 7 Purification of Resilin-CBD (SEQ ID NO: 58)

Following resilin-CBD expression, BL21 bacteria were centrifuged andlyzed as described for the other proteins. The soluble and insolublefractions were separated by centrifugation. The lysate was filtered witha syringe filter of 0.45 μm. Proteins were then loaded on to apreequilibrated Ni-NTA column and were eluted with a linear gradient of20 mM phosphate buffer (pH 7.5, 0.5 M imidazole, 0.5 M NaCl). Thefractions containing the peak that was detected at O.D. 280 nm werepooled and dialyzed three times against phosphate buffer saline (PBS) toremove the imidazole. The proteins were boiled with ×2 sampleapplication buffer (SAB) and analyzed by Coomassie-stained SDS-PAGE(FIG. 16).

Table 16 herein below summarizes the cloned resilin proteins describedherein.

TABLE 16 Number of elastic Expression Protein Sequence repeats vectorExpressed in Resilin SEQ ID NO: 17 pHis-parallel3 BL21(DE3) 56Resilin-ChBD SEQ ID NO: 17 pHis-parallel3 BL21(DE3) 55 CBD-Resilin SEQID NO: 17 pHis-parallel3 BL21(DE3) 57 Resilin-CBD SEQ ID NO: 17pHis-parallel3 BL21(DE3) 58

Example 8 Heat Resistance and Cellulose Binding Assay of Resilin-CBD(SEQ ID NO: 58)

A sample solution containing the purified resilin-CBD protein wasincubated at 85° C. for 15 minutes followed by centrifugation for 15minutes at 14,000 rpm. 50 μl of the heated protein solution was added to30 mg of cellulose powder (Sigmacell) for the purpose of cellulosebinding assay as described in Example 3. The cellulose binding assay wasalso performed with a non-heated resilin-CBD solution as control. Asshown in FIG. 17, the resilin-CBD protein displays both heat resistanceand efficient binding capacity to cellulose that was not compromised bythe heat treatment.

Example 9 Solubility of Resilin Proteins in Solutions of Different pHMaterials and Methods

There is increasing evidence that reactive oxygen species (ROS)-inducedoxidative stress resulting from enzymatic or metal-catalyzed oxidation(MCO) reactions, can highly affect protein side chains and overallcharacter. Tyrosine is one of the most ROS-sensitive residues inproteins. Its oxidation products include 3,4-dihydroxyphenylalanine(DOPA), dopamine, dopamine quinine, dityrosine (DT) and isoDT. Inaddition, DOPA is the major product of hydroxyl radical treatment oftyrosine (Ali F. E. et al., Journal of inorganic biochemistry 2004, 98,173-184). According to Ali et al (2004), MCO of tyrosine in solutions ofvarying pHs results in varying products such as dityrosine and3,4-dihydroxyphenylalanine (DOPA).

In order to use the MCO system to achieve these modifications on theresilin proteins, their stability under such pH conditions was analyzed.

Protein solutions of resilin and resilin-ChBD (pH ˜7.5) were gentlytitered with 2M HCl to pH 5.6 or pH 5.4. During the titration, 200 μlsamples, representing different pH between the starting point, and thefinal pH were collected. The samples were incubated at 4° C. for 72hours to allow for protein precipitation and then centrifuged for 15minutes at 14000 rpm. The soluble proteins were detected on aCoomassie-stained SDS-PAGE.

Results

In both cases, massive protein precipitation was observed atapproximately pH 5. As illustrated in FIG. 18, the proteins remained insolutions of pH up to 5.6 and 5.4, respectively, demonstrating the pHrange of solubility of these recombinant proteins. With thesefundamental determinations, the effect of MCO on resilin side chains canbe studied.

Example 10 Light Induced Polymerization of Resilin Proteins Products inDifferent pH Materials and Methods

Resilin and resilin-ChBD protein solutions (50 μl) at varying pH,containing 0.5 mM of Ru(bpy)3Cl₂.6H₂O and 2.5 mM of ammonium persulfate(APS) were subjected to sunlight for 10 minutes followed by proteinseparation and detection on a Coomassie-stained SDS-PAGE. Proteinsamples without Ru(bpy)3Cl₂.6H₂O and APS were used as control.

Results

In all the samples containing the Ru(bpy)3Cl₂.6H₂O and APS, highmolecular weight products were formed. Nevertheless, the pattern of theseemingly crosslinked products differed according to the pH (FIG. 19,see arrow).

Example 11 Metal-Catalyzed Polymerization of Resilin Materials andMethods

Purified resilin was dialyzed three times against either 50 mM phosphatebuffer (pH 7.5) or deionized water. Following the dialysis, the proteinswere incubated at 85° C. for 15 minutes and subsequently centrifuged for30 minutes at 10000 rpm. Generally, the polymerization was performedaccording to the MCO method reported by Kato et al (2001) (Kato Y,Kitamoto N, Kawai Y, Osawa T. (2001) The hydrogen peroxide/copper ionsystem, but not other metal-catalyzed oxidations systems, producesprotein-bound dityrosine. Free Radical Biology & Medicine, 31,(5),624-632) and Ali et al (Ali F E, Barnham K J, Barrow C J, Separovic F.(2004) Metal catalyzed oxidation of tyrosine residues by differentoxidation systems of copper/hydrogen peroxide. J Inorg Biochem.98(1):173-84). All the reactions were performed at a final volume of 250μl in 1.5 ml eppendorf tubes. The MCO polymerization was performed byadding 4 mmol H₂O₂ (1 μl of 30% H₂O₂) and 200 μM CuCl₂ (2.5 μl of 20 mMCuCl₂ dissolved in H₂O) followed by O.N. incubation at 37° C. Tubes withprotein solutions only, protein solutions with H₂O₂ only or CuCl₂ onlywere used as negative controls. The reactions were terminated by adding1 mM EDTA. Finally, the samples were boiled in ×2 SAB and were analyzedby SDS-PAGE.

Results

Polymerization was achieved in both phosphate buffer and water, asdisplayed in FIG. 20. Further analysis of these results is under way.

Example 12 Preparation of Recombinant Resilin-Cellulose WhiskerComposites Materials and Methods

His tag-purified protein solutions containing 10 mg/ml of 6H-Res-ChBD(SEQ ID NO: 55) were mounted onto a 10 kDa cutoff Vivaspin CentrifugalConcentrator (Sartorius, UK) and centrifuged at 6000 rpm to aconcentration of 100 mg/ml. At this stage, a 200 μl sample was removedand stored for later analysis, while the rest of the solution wasfurther concentrated to 200 mg/ml concentration.

6H-Res-ChBD-cellulose whiskers composites were produced by casting equalvolumes of 200 mg/ml 6H-Res-ChBD-cellulose whiskers solution andcellulose whiskers solution (prepared as describe in Bondeson D, MathewA, Oksman K. (2006) Cellulose 13:171-180) into 150 μl and 75 μl Teflonmolds resulting in final protein concentration of 100 mg/ml. 150 μl of a100 mg/ml pure 6H-Res-ChBD solution was poured into a similar mold ascontrol. Subsequently 250 μM of Ru(bpy)₃ and 2.5 mM of ammoniumpersulfate (APS) were added to each sample solution. The mixtures werehomogenized in the molds by pipeting, followed by polymerization byinduced crosslinking via exposure to a 500 W tungsten light for 5seconds.

Results

The 150 μl 6H-Res-ChBD sample (FIG. 21B—far right) and the 75 and 150 μl6H-Res-ChBD-cellulose whiskers sample composites (FIG. 21B—middle andleft, respectively) were removed from the mold and sent to DifferentialScanning calorimetry (DSC) for further analysis.

Example 13 Construction of Spider Silk-CBD Fusion Genes Materials andMethos

The spider silk (SpS) is a synthetic gene (SEQ ID NO: 23) optimized forexpression in E. coli. Its sequence is a design of 15 repeats of amonomer consensus derived from the native sequence of the spidroin 1sequence of Nephila clavipes (Accession P19837).

The SpS synthetic gene was provided in a pET30a vector, which containsan N and C terminal His tag and an Enterokinase cleavage site enablingpurification of the protein on Ni-NTA column and removal of theN-terminal His tag if desired.

Construction of SpS-CBD Fusion Genes for Expression in E. coli:

Clostridium cellulovorans CBD (CBDclos) (SEQ ID NO: 25) was fused to the3′ of the spider silk synthetic gene. The fusion gene is referred to asSpS-CBD (SEQ ID NO: 24).

PCR primers were designed in order to construct the SpS-CBD fusion geneas summarized in Table 17 herein below. The PCR primers will add anN-terminal SpeI and a C-terminal XhoI restriction sites to the CBDclosgene template.

TABLE 17 SEQ ID Tm° No. Primer name Sequence description C. 37 CBDSpeI_GACTAGTATGGCAGC Forward primer 56 for GACATCATCAATGTC of CBD160 +SpeI site 47 CBDSXhoI_ CTCGAGATCAAATGT Reverse primer 56 revTGCAGAAGTAGGATT of CBD160 + AATTATTG XhoI site

The CBDclos gene served as a PCR template for cloning of the fusiongenes. A standard PCR was performed using Ex Taq™ (Takara, MadisonWis.), which is a proof reading enzyme suitable for TA cloning. The PCRproduct was purified from a 1% agarose gel and was cloned into pGEM-TEasy vector (Promega Corporation, Madison Wis.). The presence ofSpeI-CBDclos-XhoI was verified by sequencing.

Cloning of SpS-CBD—

The SpeI-CBDclos-XhoI was cloned into SpeI and XhoI restriction sites onpET30a-SpS vector.

Construction of Spider Silk Genes Optimized for Expression in TobaccoPlants:

The synthetic dragline silk gene (GENEART GmbH Regensburg, Germany, SEQID NO: 27) is composed of a repeat unit, which was selected based on aconsensus (GPGGQGPYGPGASAAAAAAGGYGPGYGQQGPGQQGPGQQ) SEQ ID NO:26 derivedfrom the native sequence of the Arenaus diadematus ADF-3 gene (AccessionU47855). Multimers encoding this repeat were developed by the use of thecondensation method [Lewis et al., Protein Expression and Purification7, 400-406 (1996)]. The synthetic gene includes the sequence of themonomer limited by the SmaI and NaeI restriction sites, which were usedfor the development of the multimers with the aid of another uniquerestriction site (AatII) on the pUC19 vector.

At the end of the spider silk monomer sequence there is an addition ofthe 3′ non-repetitive sequence of the ADF-3 dragline gene. This sequencewas shown to contribute to the solubility of the protein [Lazaris etal., Science 295: 472-476 (2002)]. At the 5′ of the silk monomer apartial sequence of a synthetic CBDclos gene was added as describedherein below.

Construction of 6 Monomer (6mer) Spider Silk Gene:

In order to construct a 6mer spider silk gene a double digest wasperformed as follows:

-   1. Digest of the synthetic monomer (SEQ ID NO: 26) with SmaI and    AatII.-   2. Digest of the synthetic monomer (SEQ ID NO: 26) with NaeI and    AatII.    The DNA products were purified on a 1% agarose gel and the ligation    of the purified fragments yielded a 2mer spider silk gene.    Subsequently, a condensation of 2mers was performed to create a 4mer    gene and a 4mer and a 2mer were condensed to form a 6mer gene.

Construction of 6mer-CBDclos Fusion Genes:

The sequence of the CBDclos was optimized for expression in tobaccoplants. The CBD synthetic DNA was fused to the 5′ of the silk monomer.In order to construct a full length CBDclos-6mer fusion, a digest ofBclI and NcoI restriction sites on the partial CBD-6mer gene and thefull length non synthetic CBDclos was performed.

The fusion of the CBD to the 6mer gene was made in two orientations:

-   1. Two 6mer repeats were fused to the 3′ terminal end of CBDclos to    create CBDclos-SpS12 (SEQ ID NO: 28). The condensation of two 6mers    was performed as described above.-   2. CBDclos was fused in the middle of two 6mer repeats. The fusion    gene is referred to as SpS6-CBD-SpS6 (SEQ ID NO: 29). The cloning of    the two 6mers was performed by double digestion of one CBD-6mer    plasmid with SmaI and NaeI and the other with StuI.    The fragments were purified and ligated to form SpS6-CBD-SpS6.    Both CBD-12mer and SpS6-CBD-SpS6 were cloned into Rubisco's small    subunit cassette (includes regulatory elements, such as the    promoter, terminator, 5′ and 3′ untranslated regions cloned from    Chrysanthemum sp.) SEQ ID NOs: 30 and 31, on the pBINPLUS binary    vector. Another expression cassette which was used includes the Cell    signal peptide for secretion of the fusion proteins to the apoplast.    This signal was fused to the 5′ of the fusion genes before the 5′UTR    of the Rubisco's small subunit gene.    Table 18 summarizes the cloned spider silk proteins described    herein.

TABLE 18 Number of monomer Expression Protein repeats vector/tagExpressed in Spider silk (SpS) (SEQ 15 pET30a/His BL21(DE3) ID NO: 33)Spider silk-CBD (SpS- 15 pET30a/His BL21(DE3) CBD) (SEQ ID NO: 34)CBD-spider silk (CBD- 12 pBINPLUS/Cell N. tabacum- SpS12) (SEQ ID NO:SR1 28) Spider silk-CBD-spider 12 pBINPLUS N. tabacum- silk(SpS6-CBD-SpS6) SR1 (SEQ ID NO: 29)

Example 14 Expression and Purification of SpS-CBD Fusion Genes Materialsand Methods

Expression of SpS and SpS-CBDclos Proteins in E. coli: The pET30a-SpSand pET30a-SpS-CBDclos vectors were transformed into BL21(DE3) (Novagen,EMD Chemicals, Inc. CA). 5 ml of over night cultures were grown in LBmedium with 50 mg/l kanamicin at 37° C. on a rotary shaker. Thesestarters were used for inoculation of 250 to 350 ml of LB with 50 mg/lkanamycin at a ratio of 1/100 of starter to culture volume. At O.D. 600of 0.6 to 0.9, expression was induced with 1 mM IPTG. Following fourhours from induction, bacteria were harvested by centrifugation. Onepellet was divided to 50 ml aliquots for initial analysis and thepellets were stored at −80° C.

FPLC Purification of 6H-SpS and 6H-SpS-CBD:

Bacterial pellet of 300 ml was re-suspended in 5 ml of 100 mM Tris pH7.5, 0.1% Triton® X-100, Complete™ (Roche, Basel Switzerland). Bacteriawere lyzed by sonication with pulsed bursts for 5 minutes on an icebath. The soluble and bacterial precipitates were separated bycentrifugation at 15000 rpm for 10 minutes at 4° C. The soluble fractionof the proteins was filtered with a syringe filter of 0.45 μm for thepurpose of FPLC (GE, Uppsala Sweden) purification on HisTrap™ HP (GE,Uppsala Sweden) Ni-NTA 1 ml column pre-equilibrated according to theuser manual.

The purification program was run as follows:Binding buffer; 20 mM NaHPO₄, 0.5 M NaCl, 10 mM imidazoleElution buffer; 20 mM NaHPO₄, 0.5 M NaCl, 0.5 M imidazole

-   1. 5 column volumes (CV) of binding buffer at 1 ml/min.-   2. 5 ml injection of the lysate at 1 ml/min-   3. 10 CV wash with the binding buffer.-   4. linear gradient up to 500 mM imidazole for 15 minutes at 1 ml/min    with the elution buffer-   5. Equilibration with 10 CV of binding buffer at 1 ml/min.    Eluted proteins were detected at O.D. 280. 500 μl fractions were    collected and 20 μl of samples boiled with SAB were loaded on a 10%    SDS-PAGE gel.

Expression of CBD-SpS12 and SpS6-CBD-SpS6 Proteins in Tobacco Plants

Transformation of Tobacco Plants:

The binary pBINPLUS vector including the Robisco's expression cassetteand the fusion genes were introduced into A. tumefaciens strain LBA4404for plant transformation. Leaf-disc transformation was performed with N.tabacum-SR1 plants as described previously (DeBlock et al., 1984 TheEMBO Journal vol. 3 no. 8 pp. 1681-1689, 1984). More than 15 independenttobacco transformants were generated for each construct, propagated invitro and transferred to the greenhouse. The presence of the transgenewas confirmed by PCR on genomic DNA using specific primers for theRobisco's cassette terminator/promoter. T1 seeds obtained byself-pollination of transformants were harvested and selected further ongermination medium containing kanamycin (300 mg 1-1). The sterilizationtreatment was for 30 seconds in 70% ethanol followed by 5 minutes 2.5%NaOCl.

Expression of CBD-SpS12 and SpS6-CBD-SpS6 by T1 Homozygous Plants:

Protein extraction was performed by grinding 90 mg of transgenic tobaccoleaves with chilled extraction buffer (50 mM Tris-HCL pH=7.5,“complete”-protease inhibitor cocktail tablets. Roche-Cat#1697498) in atissueLyser (Retch Mixer Mill Type MM301/220-240V 50/60HZ.cat#20.741.0001). Separation of soluble and insoluble fractions wasdone by centrifugation at 15000 rpm for 10 minutes at 4° C. Soluble andinsoluble fractions were boiled with SAB.

Purification of CBD-SpS12 and SpS6-CBD-SpS6 from Transgenic TobaccoPlants:

20 mg of transgenic leaves in 40 ml purification buffer (50 mM Tris-HCLpH=7.5, 10 mM DTT, 0.5 gr cellulose Sigmacell20, PMSF 1 mM were groundin a blender till a uniform mixture was obtained. Separation of solubleand insoluble fractions was performed by centrifugation at 14000 rpm for15 minutes at 4° C. The insoluble fraction, which includes the bound CBDfusion proteins, was washed extensively twice in 30 ml extraction buffereach. The bound proteins were eluted from the cellulose pellet bysuspension in elution buffer (50 mM Tris-HCL pH=12.5, 10 mM DTT, 0.1%Triton) for 1 hour in a shaking rotor. Separation of the solublefraction, which includes the eluted CBD fusion protein, was effected bycentrifugation at 14000 RPM for 15 minutes at 4° C.

Further Purification of SpS6-CBD-SpS6:

The eluted soluble protein from the procedure detailed above wasdialyzed against 5 liter of heat stability test buffer (50 mM sodiumphosphate pH=8, 10 mM DTT) over night. Then the sample was centrifugedat 14000 RPM for 10 minutes at 4° C. The soluble protein was subjectedto heat treatment in 60-90° C. for 10 minutes, followed by 20 minutes onice, and centrifuging at a maximum speed for 10 minutes. The solubleprotein was also tested for its solubility at a wide range of pHs from8-2. The pH of the heat stability test buffer was adjusted with 2M HCLuntil the pH of the solution reached pH=2. For every pH coordinate, asample was taken for analysis and incubated at 4° C. overnight. Toseparate soluble from insoluble, the samples were centrifuged at amaximum speed for 10 minutes. The soluble proteins were boiled withSDS-PAGE sample application buffer (SAB).

Qualitative Binding Assay of Purified SpS and SpS-CBD to Cellulose:

30 mg of cellulose (Sigmacell) were added to 1.5 ml eppendorf tubes. Thematerials were washed with PBS followed by addition of 50 μl of affinitypurified protein solution. 450 μl of PBS were added to each tube to atotal reaction volume of 500 μl. The tubes were incubated under gentlespinning for 30 minutes at RT followed by centrifugation. Thesupernatant was removed (unbound fraction) and the pellets were washedfor 3 times with 500 μl of PBS. The final pellets were boiled with 50 μlof SAB. Samples of unbound fraction from each tube were also boiled withSAB. Samples were loaded on 10% SDS-PAGE gel.

Quantitative Binding Reversibility Assay of Purified CBDclos, SpS andSpS-CBDclos to Cellulose:

100 to 600 μg of SpS and SpS-CBD proteins in 500 μL PBS were adsorbed to30 mg prewashed cellulose (Sigmacell) for 30 minutes at 25° C.Desorption from the cellulose was performed, while the most concentratedprotein:cellulose mixture (600 μg+30 mg cellulose) was diluted inindividual test tubes to final protein quantity ranging from 600 to 100μg, followed by mixing for an additional 30 minutes. Aftercentrifugation at 13000 g for 10 minutes, the bound proteinconcentration was assayed by the Lowry method (The NaOH in the Lowry Asolution elutes the bound proteins from the cellulose pellet).

Results

Expression of SpS and SpS-CBDclos Proteins in E. coli:

The SpS and SpS-CBD proteins were successfully expressed in E. coli(FIG. 22A). SDS-PAGE analysis of soluble and insoluble (IB content)proteins revealed that the SpS protein product is found in the solublefraction, whereas SpS-CBD protein product is mostly found in theinsoluble inclusion bodies (IB) fraction (FIG. 22B).

FPLC Purification of 6H-SpS and 6H-SpS-CBD:

The SpS and SpS-CBD proteins were successfully purified on a Ni-NTAcolumn (FIG. 23A). The purified SpS and SpS-CBD were identified byanti-6HIS antibody (FIG. 23B). When looking at the chromatogram of thepurification on Ni-NTA (FIGS. 24A-C), a non specific protein peak can beobserved in the control run (FIG. 24A). The protein which was eluted isidentified by literature as SlyD. This doesn't interfere with the SpSand SpS-CBD purification as SlyD elutes prior to the fusion proteins(FIG. 24A lanes 5-7).

Qualitative Binding Assay of Purified 15mer and 15mer-CBD to Cellulose:

Coomassie blue staining showed that the SpS-CBD was bound to cellulose,with no apparent protein revealed by Coomassie blue in the unboundfraction (FIG. 25 lanes 5-7). The SpS is mostly found in the unboundfraction following the binding procedure (FIG. 26 lanes 2-4). The SpSprotein found in the bound fraction is nonspecifically adsorbed tocellulose. This phenomenon can be explained by the mechanism of proteinsadsorption in solid/liquid interfaces [Haynes et al, Colloids andSurfaces B, Biointerfaces. 2:517-566 (1994)] as further demonstratedbelow.

Quantitative Binding Reversibility Assay of Purified CBDclos, SpS andSpS-CBDclos to Cellulose:

Adsorption/desorption experiments are critical tests to study thereversible nature of adsorption. A reversible adsorption process isdefined if the departure from adsorption equilibrium is infinitesimallysmall, so that in the reverse process (desorption) the variablescharacterizing the state of the system return to the same values in thereverse order. Therefore in a reversible adsorption process, theascending branch (increasing concentration in the solution) and thedescending branch (decreasing concentration in the solution) of theisotherm must overlap. If the ascending and descending branches of theisotherm do not overlap, the process is defined as irreversible and thedeviation between the ascending and descending branches is defined ashysteresis [Haynes et al, Colloids and Surfaces B, Biointerfaces.2:517-566 (1994)]. The desorption experiments of CBDclos and SpS-CBDclosrevealed that a new equilibrium was established after dilution, whichwas not on the same isotherm (FIG. 26). These results prove that theascending and descending isotherms do not overlap, which is aprerequisite for irreversible binding. These results demonstrate thatunder the conditions tested, CBDclos and SpS-CBDclos display similaradsorption behavior and bind almost irreversibly to cellulose. Theresults also reveal that the ascending and descending branches of theSpS isotherm almost overlap, therefore it can be known for certain thatthe SpS adsorption to cellulose is not reversible but rather due toprotein adsorption in solid/liquid interfaces. Table 19 herein belowsummarizes the results quantitative binding reversibility assay results.

TABLE 19 Total Reverse protein Bound (μg)/10 mg cellulose binding(μg)/10 mg cellulose (μg) CBD SpS SpSr-CBD CBD SpS SpS-CBD 100 99.2114.05 94.15 474.56 3.41 408.13 150 138.67 23.28 120.92 467.57 13.02402.88 300 237.38 41.57 231.47 442.22 77.71 416.87 600 462.17 101.33419.58 462.17 101.33 419.58

Expression of CBD-SpS12 and SpS6-CBD-SpS6 by T1 Homozygous Plants:

Four homozygous T1 plants, with elevated protein expression, wereisolated:

-   1. Two plants of CBD-SpS12 number 13.7 and 13.8, which express and    secrete CBD-SpS12 to the appoplast were identified, referred to    herein as 13.7 and 13.8, respectively.-   2. Two plants of SpS6-CBD-SpS6 number 6.4 and 6.8, which express    6mer-CBD-SpS6 in the cytoplasm were identified, referred to herein    as 6.4 and 6.8, respectively.    SDS-PAGE analysis of protein extracts revealed that both CBD-SpS12    and SpS6-CBD-SpS6 bound cellulose and therefore were mostly found in    the insoluble fraction (FIGS. 27A-B). With the addition of extra    cellulose to the extraction procedure, all the soluble fraction of    the CBD fusion proteins bound cellulose.

Purification of CBD-SpS12 and SpS6-CBD-SpS6 from Transgenic TobaccoPlants:

The purification of CBD-SpS 12 and SpS6-CBD-SpS6 is based on the uniquebinding of the fusion CBD proteins to the plant's cell wall. Thisspecific binding confirms that the CBD is active and serves as the firststep of purification (FIG. 28A). CBD-containing proteins were shown tobind the cell wall and to precipitate along with the insoluble fractionsof the cell extract. The pellet was then treated with elution buffer,leading to release of CBD-containing proteins to the soluble fraction ofthis elution process (FIG. 28A, lane 6). Further purification ofSpS6-CBD-SpS6 is based on the spider silk unique heat stability andsolubility at a wide range of pHs. From SDS PAGE analysis it is clearthat the SpS6-CBD-SpS6 is heat stable and soluble at a wide range of pHs(FIG. 28B).

Example 14 Metal Catalyzed Polymerization of Spider Silk Materials andMethods

Purified SpS protein (Example 8), containing 15 tyrosine residues, wasdialyzed four times against either 50 mM phosphate buffer (pH 7.5) ordeionized water. Following the dialysis, the protein was centrifuged for10 minutes at 13000 rpm (FIG. 29, lanes 2,3,4). The polymerizationreaction was performed according to the MCO method reported by Kato etal (Kato Y, Kitamoto N, Kawai Y, Osawa T. (2001) The hydrogenperoxide/copper ion system, but not other metal-catalyzed oxidationssystems, produces protein-bound dityrosine. Free Radical Biology &Medicine, 31,(5), 624-632) and Ali et al (Ali F E, Barnham K J, Barrow CJ, Separovic F. (2004) Metal catalyzed oxidation of tyrosine residues bydifferent oxidation systems of copper/hydrogen peroxide. J InorgBiochem. 98(1):173-84). All the reactions were performed in 250 μlsolution volume in 1.5 ml eppendorf tubes. The MCO polymerization wasperformed by adding 4 mmol H₂O₂ (1 μl of 30% H₂O₂) and 200 μM CuCl₂ (2.5μl of 20 mM CuCl₂ dissolved in H₂O) followed by O.N. incubation at 37°C. Tubes with protein solution only, protein solution with H₂O₂ only orCuCl₂ only were used as negative controls. The reactions were terminatedby adding 1 mM EDTA. Finally the samples were boiled in ×2 SAB andanalyzed by Coomassie-stained SDS-PAGE.

Results

Polymerization was achieved in both phosphate buffer and water, asdisplayed in FIG. 29, lanes 3 and 7.

Example 15 Method for Preparation of Spider Silk and Cellulose WhiskersSponges with/without CBD Materials and Methods

Aqueous protein solutions (5 wt %) were mixed with cellulose whiskers ina Teflon mold. After obtaining a homogenous solution, 100% methanol wasadded to the protein-whiskers mixture to a final concentration of 15%(stirring was manually performed). The mold was placed in a −80° C.freezer for more than 1 hour. The protein-whiskers frozen solution wasfreeze-dried to generate a sponge. This method is based on Nazarov R etal. Porous 3-D scaffolds from regenerated silk fibroin.Biomacromolecules (2004): 5, 718-726.

Example 16 Preparation of Recombinant a Spider Silk-Cellulose WhiskerSponge

The purified SpS protein was dialyzed against water for 18 hours,changing the water four times (the first change after 12 hours and thefollowing three changes every two hours). After dialysis, the proteinaqueous solution was concentrated to 5 wt % (FIG. 30, lanes 2 and 4 vs.5). The concentrated SpS protein was then mixed with cellulose whiskersin a Teflon mold to yield a desired ratio of 100/0%, 30/70%, 0/100%,respectively.

Example 17 Determination of Tm of Silk-Whisker Composites Materials andMethods

Sponges, generated according to the methods described in Example 15 and16, were analyzed by differential scanning calorimetry (DSC). For eachrun, ˜5 mg of sample was used, and the thermogram was recorded from0-300° C. at a heating rate of 5° C./min, under nitrogen.

Results

The DSC analysis (FIGS. 31A-C) shows three different thermogramprofiles. In the composite spider silk-cellulose whiskers thermogram thetransition temperature peak 2 of spider silk and cellulose whiskersalone disappeared and a higher peak appeared at 243.69° C. Table 20summarizes the transition temperature peaks from DSC thermograms ofwhiskers, silk and 70% whiskers/30% silk sponges. This analysisdemonstrates that the silk-whisker combination leads to a significantincrease in whiskers transition temperature peak2. Table 20 summarizesthe transition temperature peaks from DSC thermograms of whiskers, silkand 70% whiskers/30% silk sponges.

TABLE 20 Transition temp. Tg Transition temp. peak1 (° c.) (° c.) peak2(° c.) Cellulose whiskers 93.04 — 193.71 (FIG. 2A) Silk (FIG. 2B) 81.07175.44 267.66 70% whiskers/30% silk 87.56 — 243.69 (FIG. 2C) Transitiontemp. Transition temp. Sample peak1 (° C.) peak2 (° C.) Cellulosewhiskers 93.04 193.71 Silk 81.07 175.44 (Tg) 70% whiskers/30% 87.56243.69 silk

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

What is claimed is:
 1. A composite comprising resilin and cellulose,wherein said cellulose is in the form of whiskers.
 2. A compositecomprising a fibrous polypeptide and cellulose, said fibrous polypeptidecomprising a heterologous polysaccharide binding domain, the compositebeing non-immobilized, wherein said cellulose is in the form ofwhiskers.
 3. The composite of claim 1, wherein said resilin comprises acellulose binding domain.
 4. The composite of claim 2, wherein saidfibrous polypeptide is selected from the group consisting of musselbyssus protein, resilin, silkworm silk protein, spider silk protein,collagen, elastin and fragments thereof.
 5. The composite of claim 1,further comprising an additional polypeptide being selected from thegroup consisting of mussel byssus protein, silkworm silk protein, spidersilk protein, collagen, elastin and fragments thereof.
 6. The compositeof claim 1 being crosslinked.
 7. The composite of claim 1 beingnon-crosslinked.
 8. The composite of claim 1, wherein said resilincomprises at least two repeating units of the sequence as set forth inSEQ ID NO:
 45. 9. A method of generating the composite of claim 1, themethod comprising contacting resilin with cellulose whiskers, therebygenerating the isolated composite of claim
 1. 10. The method of claim 9,further comprising crosslinking said composite following said contactingso as to form dityrosine bonds in said resilin.
 11. The method of claim10, further comprising coating said composite with an additional fibrouspolypeptide, said coating being effected following said crosslinking thecomposite.
 12. The method claim 9, further comprising binding saidresilin with an additional fibrous polypeptide prior to said contacting.